Methods of manipulating nucleic acids

ABSTRACT

Methods are provided for labeling nucleic acid molecules for use in hybridization reactions, and kits employing these methods. The level of labeling is increased by including one or more reactive modifications, such as amine-modifications, into the primers used to initiate synthesis of the nucleic acid molecule, for instance through random-primed reverse transcription. Also provided are modified random primers (such as amine-modified random primers) useful in these methods, labeling and hybridization kits comprising such primers, labeled nucleic acid molecules and mixtures of molecules, and methods for using them. Methods are also provided for amplifying a nucleic acid template contained within extremely small samples, in some cases as little as one cell. In particular embodiments, a single random primer is used for all steps of the amplification method. The nucleic acid template can either be of cellular or viral origin.

REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation in part of International PatentApplication No. PCT/US02/11656, filed Apr. 11, 2002, which in turnclaims the benefit of U.S. Provisional Application No. 60/283,423, filedApr. 11, 2001, both of which are incorporated herein by reference.

FIELD

[0002] This disclosure relates to methods of labeling nucleic acidprobes for the detection of nucleic acids molecules, for instanceproducing labeled probes for detecting hybridization signals, such asthose from a microarray.

BACKGROUND OF THE DISCLOSURE

[0003] Microarray technology involves depositing nucleic acids (thetarget) on a solid platform (e.g., a glass microscope slide or chip) ina set pattern, and hybridizing a solution of labeled, potentiallycomplementary nucleic acids (the probe) to the nueleic acid targets.This technology has been successfully applied to the simultaneousanalysis of expression of many thousands of genes and large-scale genediscovery, as well as to polymorphism screening and mapping of genomicDNA clones. Microarray technology permits quantitative gene expressionanalysis using RNA transcripts from known and unknown genes, as well asqualitative detection of, for instance, human pathogens anddisease-related genes from DNA samples.

[0004] Most applications using DNA arrays involve preparation offluorescent labeled cDNA from the mRNA of the studied organism. The cDNAprobes are then allowed to hybridize with the DNA fragments printed onthe array, and the resulting hybridization profile is then scanned by aconfocal microscope and analyzed by the appropriate software.

[0005] Two probe labeling strategies for microarray studies have beendeveloped. The most commonly used involves directly incorporatingfluorescent nucleotides (such as Cy3-dUTP and Cy5-dUTP) into cDNA probesthrough reverse transcription primed by an oligo dT primer (Duggan, etal., Nat. Genet. Suppl. 21:10-14, 1999). The optimal ratio ofdye-modified to unmodified nucleotide used is governed by twofactors: 1) that modified bases cause a deterioration in the strengthand specificity of binding of probes to their target DNAs, and 2) thatas many modified bases as possible have to be incorporated into probesto give good fluorescent signals. In practice, this trade-off limits theefficiency of probe labeling, and a large amount of starting RNA isrequired to produce labeled probe for each hybridization.

[0006] The second currently available labeling method is indirectlabeling, wherein the cDNA is synthesized in the presence ofamine-modified nucleotides (e.g., aminoallyl dUTP), and the fluorescentdyes are subsequently coupled onto the cDNA molecules by reaction withthese amine groups. The same factors that limit the efficiency of directlabeling limit the efficiency of the indirect labeling method. Becauseof these problems, even optimal labeling reactions require a largequantity of mRNA (2 μg or more) or total RNA (20 μg or more) to produceenough probe to give a good hybridization signal. So much startingmaterial is required that certain samples (such as clinical biopsies andmicrodissected cells) cannot be studied.

[0007] Recently, expensive, time consuming, multi-step procedures foramplifying and then labeling probe have been reported. These permit oneto study much smaller samples than could be studied with conventionalprobe labeling methods. They are not ideal for routine studies, however,and are not sensitive enough for single cell experiments.

[0008] Protocols and reagents for conventional probe labeling areavailable commercially, for instance from companies that providefluorescent-labeled nucleotides and kits for performing such labelingreactions (e.g., Amersham's CyScribe™ First-Strand cDNA Labeling Kit).Molecular Probes has recently released a new product line (ARES™ DNALabeling Kits), which provides methods and reagents for incorporatingaminoallyl-dUTP during the reverse transcription reaction, followed byaddition of a reactive fluorescent dye, to produce labeled cDNA forvarious uses.

[0009] However, the existing nucleic acid/probe labeling methods do notprovide good quality and high level labeling using very small amounts ofstarting nucleic acid. Therefore, there exists a need for a simplemethod of labeling nucleic acids from very small starting samples.

SUMMARY OF THE DISCLOSURE

[0010] This disclosure provides new methods for amplifying nucleic acidtemplates from very small samples, even as small as one cell. Nucleicacid templates amplified by the disclosed method can be used incombination with any method that requires amplified nucleic acid. Inaddition, the amplified nucleic acid can be labeled with any labelingmethod, such as the labeling method disclosed herein.

[0011] Also provided are methods for preparing modified nucleotideprobes, from either amplified or unamplified nucleic acid templates. Inone embodiment, the method includes the incorporation of modifiednucleic acids into random primers that are used to initiatepolymerization of a probe molecule. In another embodiment, the randomprimers include nucleotides that are modified by amine groups (such asaminoallyl moieties). In yet other embodiments, the modified nucleotidescomprise a detectable molecule, such as a fluorophore or hapten.

[0012] Kits for producing a labeled hybridization probe, using amodified random primer, or for probing an array are disclosed. Alsoprovided are kits for amplifying nucleic acid templates from very smallsamples.

[0013] The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

SEQUENCE LISTING

[0014] The nucleic and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three letter code for amino acids. Only one strandof each nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

[0015] SEQ ID NOs: 1 through 10 are several modified random primers.

[0016] SEQ ID NO: 11 is an oligo dT₍₁₅₎-T7 primer.

[0017] SEQ ID NO: 12 is a primer including the T3 promoter and a random9-mer (T3N9).

[0018] SEQ ID NO: 13 is an oligo dT₍₂₀₎-T7 primer.

[0019] SEQ ID NO: 14 is a forward HHV8 PCR primer.

[0020] SEQ ID NO: 15 is a reverse HHV8 PCR primer.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 shows the structures of amine modified nucleotidesdC-C6-NH₂ and dT-C6-NH₂, used for synthesis of amine modified randomprimers P2 and P4 (see Table 1).

[0022]FIG. 2 is a schematic representation of an example of a method forlabeling probe molecules using amine modified primers during reversetranscription of cDNA from mRNA.

[0023]FIG. 3A and 3B are scatter plots comparing the expression levelsof genes between the same (FIG. 3A) or different (FIG. 3B) startingamount of RNA sample labeled with Cy5 (X-axis) and Cy3 (Y-axis), usingamine-modified random primers (P2). The log-transformed fluorescenceintensity of each spot is shown. There was a strong correlation betweenthe signals in the two channels when either the same amount (R²=0.9901)or different amounts (R2=0.9904) were labeled.

[0024]FIG. 4 is a schematic representation of two different methods toamplify RNA. The method shown on the left uses random hexamers andT7-oligo dT primers for the second and subsequent rounds of cDNAsynthesis. The method shown on the right uses a T3N9 primer for everyround of cDNA synthesis except the first.

[0025]FIG. 5 is a series of scatter plot analyses showing thereliability of a disclosed labeling method throughout multiple rounds ofamplification of RNA amplified with the T3N9 primer in cDNA microarraystudies. These plots show quantification of the log of the fluorescentsignal intensity of (A) total RNA versus RNA from first roundamplification, (B) RNA from first round amplification versus RNA fromsecond round amplification, (C) RNA from second round amplificationversus RNA from third round amplification, (D) RNA from third roundamplification versus RNA from fourth round amplification, (E) RNA fromfirst round amplification versus RNA from third round amplificationusing T3N9 primers, and (F) RNA from first round amplification versusRNA from third round amplification using random hexamers and T7-oligo dTprimers.

[0026]FIG. 6 is a schematic representation of a method to amplifynucleic acid template. The method uses a T3N9 primer for every round ofcDNA synthesis and amplification. Though illustrated with total RNA, themethod works equally well for DNA templates or starting material thatincludes both DNA and RNA.

[0027]FIG. 7 is a series of DNA microarrays and a PCR analysisdemonstrating that multiple amplification steps, using the T3N9 primerin combination with microarray detection, can be used to assay viral DNAwith a sensitivity and specificity better than PCR. The DNA microarrayin FIG. 7A has 88 open reading frames of HHV8 virus, as well as 100human house-keeping genes, printed in duplicate. Varying amounts ofgenomic DNA from HHV8 infected BCBL-1 cells were transcribed inreactions primed with random nine-mers having a T3 RNA polymeraserecognition sequence of their 5′ ends. T3 polymerase was used for theamplification step. The resulting RNA was labeled as described inExample 6, below, and hybridized to the DNA arrays. PCR products of aPCR amplification of various amounts of an HHV8 genomic DNA fragment,derived from the same template used in the amplification methoddemonstrated in FIG. 7A, were separated on a gel and are shown in FIG.7B.

DETAILED DESCRIPTION

[0028] I. Abbreviations

[0029] aa-dNTP: aminoallyl-deoxy-nucleoside triphosphate

[0030] aRNA: amplified RNA

[0031] asRNA: antisense RNA

[0032] CDs: coding sequences

[0033] cRNA: copy RNA

[0034] dN₆: random hexamer

[0035] dNTP: deoxy-nucleoside triphosphate

[0036] dA-C₆-NH₂: amino allyl modified adenine

[0037] dC-C₆-NH₂: amino allyl modified cytosine

[0038] dG-C₆-NH₂: amino allyl modified guanine

[0039] dT-C₆-NH₂: amino allyl modified thymine

[0040] FISH: fluorescent in situ hybridization

[0041] HHV8: human herpes virus-8

[0042] ORF: open reading frame

[0043] PCR: polymerase chain reaction

[0044] RT: reverse transcription (transcriptase)

[0045] SSII RT: Superscript II reverse transcriptase

[0046] T3N9: primer including the T3 promoter and a random 9-mer

[0047] II. Terms

[0048] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology. a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0049] In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

[0050] Amplification: An increase in the amount of (number of copies of)nucleic acid sequence, wherein the increased sequence is the same as orcomplementary to the existing nucleic acid template. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization (annealing) of the primers to nucleic acid template in thesample. The primers are extended under suitable conditions (thoughnucleic acid polymerization). If additional copies of the nucleic acidare desired, the first copy is dissociated from the template, andadditional copies of the primers (usually contained in the same reactionmixture) are annealed to the template, extended, and dissociatedrepeatedly to amplify the desired number of copies of the nucleic acid.

[0051] The products of amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,hybridization, ligation, and/or nucleic acid sequencing, using standardtechniques.

[0052] Other examples of in vitro amplification techniques includereverse-transcription PCR (RT-PCR), strand displacement amplification(see U.S. Pat. No. 5,744,311); transcription-free isothermalamplification (see U.S. Pat. No. 6,033,881); repair chain reactionamplification (see WO 90/01069); ligase chain reaction amplification(see EP-A-320 308); gap filling ligase chain reaction amplification (seeU.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S.Pat. No. 6,027,889); and NASBA™ RNA transcription-free amplification(see U.S. Pat. No. 6,025,134).

[0053] Antisense RNA (asRNA): A molecule of RNA complementary to a sense(encoding) nucleic acid molecule. Often, asRNA is constructed bytranscribing antisense strand RNA from a cDNA molecule.

[0054] Array: An arrangement of molecules, particularly biologicalmacromolecules (such as polypeptides or nucleic acids) in addressablelocations on a substrate. The array may be regular (arranged in uniformrows and columns, for instance) or irregular. The number of addressablelocations on the array can vary, for example from a few (such as three)to more than 50, 100, 200, 500, 1000, 10,000, or more. A “microarray” isan array that is miniaturized so as to require microscopic examination,or other magnification, for evaluation.

[0055] Within an array, each arrayed molecule is addressable, in thatits location can be reliably and consistently determined within the atleast two dimensions of the array surface. In ordered arrays thelocation of each molecule sample can be assigned to the sample at thetime when it is spotted onto the array surface, and a key may beprovided in order to correlate each location with the appropriatetarget. Often, ordered arrays are arranged in a symmetrical gridpattern, but samples could be arranged in other patterns (e.g., inradially distributed lines, spiral lines, or ordered clusters).Addressable arrays are computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation (such as hybridization or binding data, including forinstance signal intensity). In some examples of computer readableformats, the individual “spots” on the array surface will be arrangedregularly in a pattern (e.g., a Cartesian grid pattern) that can becorrelated to address information by a computer.

[0056] The sample application “spot” on an array may assume manydifferent shapes. Thus, though the term “spot” is used, it refersgenerally to a localized deposit of nucleic acid, and is not limited toa round or substantially round region. For instance, substantiallysquare regions of mixture application can be used with arraysencompassed herein, as can be regions that are substantially rectangular(such as a slot blot-type application), or triangular, oval, orirregular. The shape of the array substrate itself is also immaterial,though it is usually substantially flat and may be rectangular or squarein general shape.

[0057] Binding or stable binding: An oligonucleotide binds or stablybinds to a target nucleic acid if a sufficient amount of theoligonucleotide forms base pairs or is hybridized to its target nucleicacid, to permit detection of that binding. Binding can be detected byeither physical or functional properties of the target:oligonucleotidecomplex. Binding between a target and an oligonucleotide can be detectedby any procedure known to one skilled in the art, including bothfunctional and physical binding assays. Binding may be detectedfunctionally by determining whether binding has an observable effectupon a biosynthetic process such as expression of a coding sequence, DNAreplication, transcription, amplification and the like.

[0058] Physical methods of detecting the binding of complementarystrands of DNA or RNA are well known in the art, and include suchmethods as DNase I or chemical footprinting, gel shift and affinitycleavage assays, Northern blotting, dot blotting and light absorptiondetection procedures. For example, one method that is widely used,because it is so simple and reliable, involves observing a change inlight absorption of a solution containing an oligonucleotide (or ananalog) and a target nucleic acid at 220 to 300 nm as the temperature isslowly increased. If the oligonucleotide or analog has bound to itstarget, there is a sudden increase in absorption at a characteristictemperature as the oligonucleotide (or analog) and target disassociatefrom each other, or melt.

[0059] The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. A higher (T_(m))means a stronger or more stable complex relative to a complex with alower (T_(m)).

[0060] cDNA (complementary DNA): A piece of DNA lacking internal,non-coding segments (introns) and transcriptional regulatory sequences.cDNA may also contain untranslated regions (UTRs) that are responsiblefor translational control in the corresponding RNA molecule. cDNA isusually synthesized in the laboratory by reverse transcription frommessenger RNA extracted from cells or other samples.

[0061] Complementarity and percentage complementarity: Molecules withcomplementary nucleic acids form a stable duplex or triplex when thestrands bind, (hybridize, anneal), to each other by formingWatson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable bindingoccurs when an oligonucleotide remains detectably bound to a targetnucleic acid sequence under the required conditions.

[0062] Complementarity is the degree to which bases in one nucleic acidstrand base pair with the bases in a second nucleic acid strand.Complementarity is conveniently described by percentage, i.e. theproportion of nucleotides that form base pairs between two strands orwithin a specific region or domain of two strands. For example, if 10nucleotides of a 15-nucleotide oligonucleotide form base pairs with atargeted region of a DNA molecule, that oligonucleotide is said to have66.67% complementarity to the region of DNA targeted.

[0063] A thorough treatment of the qualitative and quantitativeconsiderations involved in establishing binding conditions that allowone skilled in the art to design appropriate oligonucleotides for useunder the desired conditions is provided by Beltz et al. Methods Enzymol100:266-285, 1983, and by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0064] Coupling: As used herein, the term “coupling” refers to thechemical reaction of a nucleotide, such as a modified nucleotide, with adetectable molecule, such as a hapten or label (e.g., a fluorophore). Byway of example, disclosed embodiments of coupling reactions may bereactions between a nucleophile (functional group) and an electrophile,i.e., an electron poor reactive group. The coupling reaction may befacilitated by using an activating moiety to activate the electrophileto nucleophilic coupling. The activating group also usually is a leavinggroup. The nucleophile can be on either the nucleotide or on thedetectable molecule, so long as the pair of reactants (nucleotide anddetectable molecule) are capable of reacting with each other. Manyembodiments have the nucleophile provided by the nucleotide.

[0065] Examples of reactions that may occur between the nucleophile andthe electron poor reactive group include (in no particular order), butare not limited to, a Grignard reaction, a Wittig reaction, acondensation (such as an aldol condensation), a Mitsunobu reaction,formation of a Schiff base, and so forth.

[0066] Representative examples of nucleophilic functional groups includeamines (—NH₂), —NHR (where R is aliphatic, e.g., an alkyl group),alcohols (—OH), thiols (—SH), acido-acetates, alkyl lithium components,and so forth. Hydrogen-bearing compounds also can be deprotonated tofacilitate the coupling reaction. Additional examples of functionalgroups will be apparent to one of ordinary skill in the art.

[0067] Representative examples of leaving groups include halides(including F, Cl, and I), sulfonates, phosphates, DCC, EDC, imidazole,DMAP, DMF/acid chloride, and so forth. Further leaving groups arelisted, for instance, in U.S. Pat. No. 5,268,486, and includeisothiocyanate, isocyanate, monochlorotriazine, dichlortriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituteddiazine, maleimide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenyl, azide, 3-(2-pridyl dithio)-proprionamide,glyoxal and aldehyde. Additional examples of leaving groups will beapparent to one of ordinary skill in the art.

[0068] Specific examples of coupling reactions between aminoallylnucleotides and fluorophores and haptens are illustrated in Nimmakayaluet al. (BioTechniques 28:518-522, 2000). Further specific examples arepresented herein.

[0069] Fluorophore: A chemical compound, which when excited by exposureto a particular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength than that to which it was exposed.Fluorophores can be described in terms of their emission profile, or“color.” Green fluorophores, for example Cy3, FITC, and Oregon Green,are characterized by their emission at wavelengths generally in therange of 515-540 λ. Red fluorophores, for example Texas Red, Cy5 andtetramethylrhodamine, are characterized by their emission at wavelengthsgenerally in the range of 590-690 λ.

[0070] Encompassed by the term “fluorophore” as it is used herein areluminescent molecules, which are chemical compounds which do not requireexposure to a particular wavelength of light to fluoresce; luminescentcompounds naturally fluoresce. Therefore, the use of luminescent signalseliminates the need for an external source of electromagnetic radiation,such as a laser. An example of a luminescent molecule includes, but isnot limited to, aequorin (Tsien, 1998, Ann. Rev. Biochem. 67:509).

[0071] Examples of fluorophores are provided in U.S. Pat. No. 5,866,366.These include: 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid, acridine and derivatives such as acridine and acridineisothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide,Brilliant Yellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron .RTM. Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

[0072] Other fluorophores include thiol-reactive europium chelates thatemit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem.248:216-227, 1997; J Biol. Chem. 274:3315-3322, 1999).

[0073] Other fluorophores include cyanine, merocyanine, styryl, andoxonyl compounds, such as those disclosed in U.S. Pat. Nos. 5,268,486;5,486,616; 5,627,027; 5,569,587; and 5,569,766, and in published PCTpatent application no. US98/00475, each of which is incorporated hereinby reference. Specific examples of fluorophores disclosed in one or moreof these patent documents include Cy3 and Cy5, for instance.

[0074] Other fluorophores include GFP, Lissamine™, diethylaminocoumarin,fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamineand xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.,herein incorporated by reference) and derivatives thereof. Otherfluorophores are known to those skilled in the art, for example thoseavailable from Molecular Probes (Eugene, Oreg.).

[0075] Particularly useful fluorophores have the ability to be attachedto (coupled with) a nucleotide, such as a modified nucleotide, aresubstantially stable against photobleaching, and have high quantumefficiency.

[0076] High throughput genomics: Application of genomic or genetic dataor analysis techniques that use microarrays or other genomictechnologies to rapidly identify large numbers of genes or proteins, ordistinguish their structure, expression or function from normal orabnormal cells or tissues.

[0077] Human Cells: Cells obtained from a member of the species Homosapiens. The cells can be obtained from any source, for exampleperipheral blood, urine, saliva, tissue biopsy, surgical specimen,amniocentesis samples and autopsy material. From these cells, genomicDNA, cDNA, mRNA, RNA, and/or protein can be isolated.

[0078] Hybridization: Oligonucleotides (and oligonucleotide analogs)hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary bases.Generally, nucleic acid consists of nitrogenous bases that are eitherpyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines(adenine (A) and guanine (G)). These nitrogenous bases form hydrogenbonds between a pyrimidine and a purine, and the bonding of thepyrimidine to the purine is referred to as “base pairing.” Morespecifically, A will hydrogen bond to T or U, and G will bond to C.

[0079] Hybridization conditions resulting in particular degrees ofstringency will vary depending upon the nature of the hybridizationmethod of choice and the composition and length of the hybridizingnucleic acid sequences. Generally, the temperature of hybridization andthe ionic strength (especially the Na⁺ concentration) of thehybridization buffer will determine the stringency of hybridization,though waste times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11, hereinincorporated by reference.

[0080] For purposes of the present invention, “stringent conditions”encompass conditions under which hybridization will only occur if thereis less than 25% mismatch between the hybridization molecule and thetarget sequence. “Stringent conditions” may be broken down intoparticular levels of stringency for more precise definition. Thus, asused herein, “moderate stringency” conditions are those under whichmolecules with more than 25% sequence mismatch will not hybridize;conditions of “medium stringency” are those under which molecules withmore than 15% mismatch will not hybridize, and conditions of “highstringency” are those under which sequences with more than 10% mismatchwill not hybridize. Conditions of “very high stringency” are those underwhich sequences with more than 6% mismatch will not hybridize.

[0081] Nucleotide: “Nucleotide” includes, but is not limited to, amonomer that includes a base linked to a sugar, such as a pyrimidine,purine or synthetic analogs thereof, or a base linked to an amino acid,as in a peptide nucleic acid (PNA). A nucleotide is one monomer in anoligonucleotide/polynucleotide. A nucleotide sequence refers to thesequence of bases in an oligonucleotide/polynucleotide.

[0082] The major nucleotides of DNA are deoxyadenosine 5′-triphosphate(dATP or A), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U). Inosine is also a basethat can be integrated into DNA or RNA in a nucleotide (dITP or ITP,respectively).

[0083] Modified nucleotide (modified nucleoside triphosphate): Amodified nucleotide is a nucleotide that has been modified, for examplea nucleotide to which a chemical moiety has been added, usually one thatgives an additional functionality to the modified nucleotide. Generally,the modification comprises a functional group or a leaving group, andpermits coupling of the nucleotide to a detectable molecule, such as afluorophore or hapten. In other embodiments, an alteration in thestructure of the nucleotide or a deletion of an atom can make thenucleotide reactive with a detectable label.

[0084] For instance, one specific class of modifications are those thatadd a reactive amine group to the nucleotide; an aminoallyl group is onesuch amine modification. Amine groups are reactive with a wide spectrumof other chemical groups, which will be known to one of ordinary skillin the art. By way of example, amine groups are reactive withintermediate N-hydroxysuccinimide (NHS) esters, such as those on NHSester cyanine dyes. Amine groups also can be reacted with peptidemolecules (such as antigenic fragments or antibody or antibody fragment)or biotin (for instance, to which a fluorescent dye can then becoupled), for instance. Examples of amine-reactive fluorophores that canbe coupled to amine modified-nucleotides include, but are not limitedto, fluorescein, BODIPY, rhodamine, Texas Red, cyanine dyes, and theirderivatives. Reaction of amine-reactive fluorophores usually proceeds atpH values in the range of pH 7-10.

[0085] Alternatively, thiol-reactive fluorophores can be used togenerate a fluorescently-labeled nucleotide or oligonucleotide. Thus,also contemplated herein are nucleotides (and oligonucleotides)containing a thiol group as its modification. Reaction of fluors withthiols usually proceeds rapidly at or below room temperature (RT) in thephysiological pH range (pH 6.5-8.0) to yield chemically stablethioesters. Examples of thiol-reactive fluorophores include, but are notlimited to: fluorescein, BODIPY, cumarin, rhodamine, Texas Red and theirderivatives.

[0086] Other functional groups that can be added to a nucleotide to makea modified nucleotide include alcohols and carboxylic acids. Thesereactive functional groups also can be used to couple a fluorophore tothe nucleotide or oligonucleotide.

[0087] In particular embodiments, fluorescently-labelednucleotides/oligonucleotides have a high fluorescence yield, and retainthe critical features of the nucleotide/oligonucleotide, primarily theability to bind to a complementary strand of a nucleic acid molecule andprime a polymerizing reaction.

[0088] The term also include nucleotides containing modified bases,modified sugar moieties and modified phosphate backbones, for example asdescribed in U.S. Pat. No. 5,866,336 to Nazarenko et al. (hereinincorporated by reference).

[0089] Examples of modified base moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N˜6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.

[0090] Examples of modified sugar moieties which may be used to modifynucleotides at any position on its structure include, but are notlimited to: arabinose, 2-fluoroarabinose, xylose, and hexose, or amodified component of the phosphate backbone, such as phosphorothioate,a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

[0091] Also included in the term “modified nucleotide” are branchednucleotides bearing more than one modification. Examples of branchednucleotides are disclosed, for instance, in Horn and Urdea (Nuc. AcidsRes. 17:6959-6967, 1989) and Nelson et al. (Nuc. Acids Res.17:7179-7186, 1989), incorporated herein by reference. The inclusion ofbranched modified nucleotides in modified random primers disclosedherein can provide even higher levels of labeling, since each branchedmodified nucleotide can accept more than one detectable molecule in acoupling reaction (or series of such reactions), one at eachmodification.

[0092] Specific examples of modified nucleotides, and oligonucleotidescomprising such modified nucleotides, are provided in U.S. Pat. Nos.4,605,735; 4,667,025; and 4,489,336, for instance, which patents areincorporated herein by reference.

[0093] In certain embodiments, modifications to nucleotides allow forincorporation of the nucleotide into a growing nucleic acid chain, forinstance through in vitro chemical synthesis (e.g., by phosphoramiditesynthesis).

[0094] Oligonucleotide: An oligonucleotide is a plurality of nucleotidesjoined by phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to compoundsthat function similarly to oligonucleotides but have non-naturallyoccurring portions. For example, oligonucleotide analogs can containnon-naturally occurring portions, such as altered sugar moieties orinter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.Functional analogs of naturally occurring polynucleotides can bind toRNA or DNA, and include peptide nucleic acid (PNA) molecules.

[0095] A modified oligonucleotide (or modified nucleic acid molecule) isone that comprises at least one modified nucleotide. Modifiedoligonucleotides may be mono-modified (i.e., carrying only one modifiednucleotide) or poly-modified (carrying more than one modifiednucleotide, either more than one of a single type or one or more each ofmultiple types). The primer described herein as “P2” is an example of amono-modified oligonucleotide. The primer described herein as “P4” is anexample of a poly-modified oligonucleotide.

[0096] Particular oligonucleotides and modified oligonucleotide caninclude linear sequences up to about 200 nucleotides in length, forexample a sequence (such as DNA or RNA) that is at least 6 bases, forexample at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200bases long, or from about 6 to about 50 bases, for example about 8-25bases, such as 8, 12, 15 or 26 bases.

[0097] Peptide Nucleic Acid (PNA): An oligonucleotide analog with abackbone comprised of monomers coupled by amide (peptide) bonds, such asamino acid monomers joined by peptide bonds.

[0098] Polymerization: Synthesis of a new nucleic acid chain(oligonucleotide or polynucleotide) by adding nucleotides to thehydroxyl group at the 3′-end of a pre-existing RNA or DNA primer using apre-existing DNA strand as the template. Polymerization usually ismediated by an enzyme such as a DNA or RNA polymerase. Specific examplesof polymerases include the large proteolytic fragment of the DNApolymerase I of the bacterium E. coli (usually referred to as Kleenexpolymerase), E. coli DNA polymerase I, and bacteriophage T7 DNApolymerase. Polymerization of a DNA strand complementary to an RNAtemplate (e.g., a cDNA complementary to a mRNA) can be carried out usingreverse transcriptase (in a reverse transcription reaction).

[0099] For in vitro polymerization reactions, it is necessary to provideto the assay mixture an amount of required cofactors such as M⁺⁺, anddATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP or other nucleosidetriphosphates, in sufficient quantity to support the degree ofamplification desired. The amounts of deoxyribonucleotide triphosphatessubstrates required for polymerizing reactions are well known to thoseof ordinary skill in the art. Nucleoside triphosphate analogues ormodified nucleoside triphosphates can be substituted or added to thosespecified above.

[0100] Primer: Primers are relatively short nucleic acid molecules,usually DNA oligonucleotides six nucleotides or more in length. Primerscan be annealed to a complementary target DNA strand (“priming”) bynucleic acid hybridization to form a hybrid between the primer and thetarget DNA strand, and then the primer extended along the target DNAstrand by a nucleic acid polymerase enzyme. Pairs of primers can be usedfor amplification of a nucleic acid sequence, e.g., by nucleic-acidamplification methods known in to those of ordinary skill in the art.

[0101] A primer is usually single stranded, which may increase theefficiency of its annealing to a template and subsequent polymerization.However, primers also may be double stranded. A double stranded primercan be treated to separate the two strands, for instance before beingused to prime a polymerization reaction (see for example, Nucleic AcidHybridization. A Practical Approach. Hames and Higgins, eds., IRL Press,Washington, 1985). By way of example, a double stranded primer can beheated to about 90°-100° C. for about 1 to 10 minutes.

[0102] Probe: A probe comprises an isolated nucleic acid attached to adetectable label or other reporter molecule, or a mixture of suchnucleic acids; also referred to as a labeled probe or labeled primer.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes. A modified probe is a probe that contains at least onemodified nucleotide residue, e.g., at least one aminoallyl-dUTP forinstance.

[0103] Probe standard: A probe molecule for use as a control inanalyzing an array. Positive probe standards include any probes that areknown to interact with at least one of the nucleic acids of the array,which may be found in certain spots, or in all spots on the array, eachspot containing a mixture (e.g., a different mixture) of nucleic acidmolecules. Negative probe standards include any probes known not tointeract with any nucleic acid sequence contained in at least onemixture of nucleic acids of the array.

[0104] Such a control probe sequence could, for instance, be designed tohybridize with a so-called “housekeeping” gene, which is known to orsuspected of maintaining a relatively constant expression level (or atleast known to be expressed) in a plurality of cells, tissues, orconditions. Many of such “housekeeping” genes are well known; specificexamples include histones, β-actin, or ribosomal subunits (either mRNAencoding for ribosomal proteins or rRNAs). Housekeeping genes can bespecific for the cell type being assayed, or the species or Kingdom fromwhich sample nucleic acid mixtures have been produced. For instance,ribulose bis-phosphate carboxylase oxygenase (RuBisCO), an enzymeinvolved in plant metabolism, may provide useful positive control probesfor use with arrays if the nucleic acid mixtures arrayed have beenderived from plant cells or tissues. Likewise, probes from the RuBisCOsequence (or any other plant-specific sequence) could provide goodnegative controls for gene profiling array spots that includeanimal-derived samples.

[0105] In some instances, as in certain of the kits that are providedherein, a probe standard will be supplied that is unlabeled. Suchunlabeled probe standards can be used in a labeling reaction as astandard for comparing labeling efficiency of the test probe that isbeing studied. In some embodiments, labeled probe standards will beprovided in the kits.

[0106] Probing: As used herein, the term “probing” refers to incubatingan array with a probe molecule (usually in solution) in order todetermine whether the probe molecule will hybridize to moleculesimmobilized on the array. Synonyms include “interrogating,”“challenging,” “screening” and “assaying” an array. Thus, an array issaid to be “probed” or “assayed” or “challenged” when it is incubatedwith (hybridized to) a probe molecule.

[0107] Purified: The term purified does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiednucleic acid preparation is one in which the specified protein is moreenriched than the nucleic acid is in its generative environment, forinstance within a cell or in a biochemical reaction chamber. Apreparation of substantially pure nucleic acid may be purified such thatthe desired nucleic acid represents at least 50% of the total nucleicacid content of the preparation. In certain embodiments, a substantiallypure nucleic acid will represent at least 60%, at least 70%, at least80%, at least 85%, at least 90%, or at least 95% or more of the totalnucleic acid content of the preparation.

[0108] Random Primer: An primer with a random sequence (see, forinstance, U.S. Pat. Nos. 5,043,272 and 5,106,727, incorporated herein byreference).

[0109] “Random” sequence means that the positions of alignment andbinding (annealing) of the primers to a template nucleic acid moleculeare substantially indeterminate with respect to the template underconditions wherein the primers are used to initiate polymerization of acomplementary nucleic acid. Methods for estimating the frequency atwhich an oligonucleotide of a certain sequence will appear in a nucleicacid polymer are described in Volinia et al. (Comp. App. Biosci. 5:33-40, 1989).

[0110] The sequences of random primers may not be random in the absolutemathematic sense. For instance, chemically synthesized random primerswill be random to the extent that physical and chemical efficiencies ofthe synthetic procedure will allow, and based on the method ofsynthesis. Random primers derived from natural sources (e.g., throughdigestion of an existing polynucleotide) may be less random, due tofavored arrangements of bases in the source organism. Oligonucleotideshaving defined sequences may satisfy the definition of random if theconditions of their use cause the locations of their apposition to thetemplate to be indeterminate. Also, random primers may be “random” onlyover a portion of their length, in that one residue within the primersequence, or a portion of the sequence, can be identified and definedprior to synthesis of the primer. Thus, any primer type is defined to berandom so long as the positions along the template nucleic acid strandat which primed nucleic acid extension occurs is largely indeterminate.

[0111] Random primers may be generated using available oligonucleotidesynthesis procedures; randomness of the sequence may be introduced byproviding a mixture of nucleic acid residues in the reaction mixture atone or more addition steps (to produce a mixture of oligonucleotideswith random sequence). Thus, a random primer can be generated bysequentially incorporating nucleic acid residues from a mixture of 25%of each of dATP, dCTP, dGTP, and dTTP, to form an oligonucleotide. Otherratios of dNTPs can be used (e.g., more or less of any one dNTP, withthe other proportions adapted so the whole amount is 100%).

[0112] The term “random primer” specifically includes a collection ofindividual oligonucleotides of different sequences, for instance whichcan be indicated by the generic formula 5′-XXXXX-3′, wherein Xrepresents a nucleotide residue that was added to the oligonucleotidefrom a mixture of a definable percentage of each dNTP. For instance, ifthe mixture contained 25% each of dATP, dCTP, dGTP, and dTTP, theindicated primer would contain a mixture of oligonucleotides that have aroughly 25% average chance of having A, C, G, or T at each position.

[0113] Recombinant: A recombinant nucleic acid is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination can be accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

[0114] RNA: A typically linear polymer of ribonucleic acid monomers,linked by phosphodiester bonds. Naturally occurring RNA molecules fallinto three general classes, messenger (mRNA, which encodes proteins),ribosomal (rRNA, components of ribosomes), and transfer (tRNA, moleculesresponsible for transferring amino acid monomers to the ribosome duringprotein synthesis). Messenger RNA includes heteronuclear (hnRNA) andmembrane-associated polysomal RNA (attached to the rough endoplasmicreticulum). Total RNA refers to a heterogeneous mixture of all types ofRNA molecules.

[0115] Sample: Includes biological samples such as those derived from ahuman or other animal source (for example, blood, stool, sera, urine,saliva, tears, biopsy samples, histology tissue samples, cellularsmears, moles, warts, etc.); bacterial or viral preparations; cellcultures; forensic samples; agricultural products; waste or drinkingwater; milk or other processed foodstuff; air; and so forth. Samplescontaining a small number of cells can be acquired by any one of anumber of methods, such as fine needle aspiration, micro-dissection,biopsy, tissue scrapes, or laser capture micro-dissection. Samples canalso be diluted to a level where they contain as few as 100 cells, 10cells or even as few as 1 cell in a sample.

[0116] Stripping: Bound probe molecules can be stripped from an array,for instance a cDNA array, in order to use the same array for anotherprobe interaction analysis (e.g., to determine gene expression level ina different cell sample). Any process that will remove substantially allof the prior probe molecule from the array, without also significantlyremoving the immobilized nucleic acid targets of the array, can be used.By way of example only, one method for stripping an array is by boilingit in stripping buffer (e.g., very low or no salt with 0.1% SDS), forinstance for about an hour or more. The stripped array may be washed,for instance in an equilibrating or low stringency buffer, prior toincubation with another probe molecule.

[0117] Where a stripability enhancer (such as the nucleotide analog ofthe StripAble™ and Strip-EZ™ system from Ambion (Austin, Tex.)) is used,the procedures provided by the manufacturer for use with this productprovide a good starting point for tailoring probing and strippingconditions for use with arrays. Addition of stripability enhancers toprobes for use with arrays is optional.

[0118] Subject: Living, multicellular vertebrate organisms, a categorythat includes both human and veterinary subjects for example, mammals,birds and primates.

[0119] Template: A nucleic acid polymer that can serve as a substratefor the synthesis of a complementary nucleic acid strand. Nucleic acidtemplates may be in a double-stranded or single-stranded form. If thenucleic acid is double-stranded at the start of the polymerizationreaction, it may be treated to denature the two strands into asingle-stranded, or partially single-stranded, form. Methods are knownto render double-stranded nucleic acids into single-stranded, orpartially single-stranded, forms, such as by heating to about 90°-100°C. for about 1 to 10 minutes, or by alkali treatment, such as at a pH of12 or greater.

[0120] A template nucleic acid molecule may be either DNA or RNA and maybe either homologous to the source or heterologous to the source of thesample in which it is contained, or both. For example, amplification ofa template in human tissue sample infected with a virus may result inamplification of both viral and human sequences. An example of such avirus is human herpes virus-8 (HHV8).

[0121] Nucleic acid synthesis (polymerization) in a “template dependentmanner” refers to synthesis wherein the sequence of the newlysynthesized strand of nucleic acid is essentially dictated bycomplementary base pairing to the sequence of a template nucleic acidstrand.

[0122] In some embodiments, a template nucleic acid may be amplifiedprior to using it to produce a nucleic acid probe using the modifiedrandom primers provided herein. For instance, an amplified template canbe produced by amplifying (through one or more rounds of amplification)mRNA molecules. Examples of methods for amplifying mRNAs are describedin Examples 4 and 5. In certain embodiments, it is beneficial if theamplification of the template molecule is such that the amplifiedtemplate reflects the relative abundance of the sequences found in theoriginal template molecules. See also Wang et al. (Nature Biotech.18:457-459, 2000), co-assigned U.S. provisional patent application No.60/192,700, filed Mar. 28, 2000, and the related PCT application (No.US01/09993), filed Mar. 28, 2001, each of which is incorporated hereinby reference.

[0123] Unless otherwise explained, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise. Hence“comprising A or B” means including A, or B, or A and B. “Comprising”means “including.” It is further to be understood that all base sizes oramino acid sizes, and all molecular weight or molecular mass values,given for nucleic acids or polypeptides, are approximate and areprovided for descriptive purposes. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

[0124] III. Description of Several Specific Embodiments

[0125] New methods are disclosed for amplifying nucleic acid templatesand for preparing modified nucleotide probes, from either amplified orunamplified nucleic acid templates.

[0126] The disclosure, in some embodiments, provides methods foramplifying a nucleic acid template where the nucleic acid templatecontacts a primer under conditions sufficient to permit base-specifichybridization between the template and the primer and under conditionssuitable for amplification of the nucleic acid template. In someembodiments, the primer includes a T3 promoter and a random 9-mer (T3N9,SEQ ID NO: 12) and the primer is used for at least one round of cDNAsynthesis. Optionally, the amplified nucleic acid template is labeled.In one embodiment, the amplified nucleic acid template is labeled usingan amine-modified random primer that contains at least one aminoallyldNTP residue known to interact with an amine-reactive fluorescent label.

[0127] One embodiment is a method of producing a modified nucleic acidprobe, which method includes contacting a nucleic acid template with amodified random primer under conditions sufficient to permitbase-specific hybridization between the template and the primer, andpolymerizing a nucleic acid molecule complementary to a nucleic acidsequence in the template, thereby incorporating at least one modifiedoligonucleotide primer into the complementary nucleic acid, to produce amodified nucleic acid probe. The modified random oligonucleotide primermay comprise, for instance, an amine-modified dNTP or alabel-substituted dNTP.

[0128] This disclosure in other embodiments provides methods forlabeling nucleic acid molecules, such as modified nucleic acidmolecules, suitable for hybridization reactions. The starting materialfor the labeling reaction can be minimal, for example a small number ofcells. In some embodiments, the amount of starting material contains aslittle as 1-2 μg of total RNA. In certain embodiments, particularlythose comprising an amplification, the amount of starting material maycontain as little as about 50 pg to about 100 pg of total RNA. In someembodiments, the starting material contains ribosomal RNA, messengerRNA, transfer RNA or mixtures these. In some embodiments, the nucleicacid starting material is DNA rather than RNA.

[0129] In some embodiments, the starting material is a small number ofcells, for instance as few as one cell. Provided methods enableamplifying nucleic acid templates contained within extremely smallsamples, including fine needle aspirates, tumor biopsies, tissuescrapes, laser-captured cells, and so forth, and thus enable genomicanalysis of these samples using microarray and other high-throughputsystems. In some embodiments, the starting material is less than about10 cells, less than about 100 cells, or less than about 1000 cells. Inanother embodiment, the starting material is about 10 cells. In yetanother embodiment, the starting material is about one cell. In anotherembodiment, the starting material is one cell. In some embodiments, thenucleic acid template is derived from a cell infected with a virus. Thevirus can be an RNA virus or a DNA virus. An example of a DNA virus ishuman herpes virus-8.

[0130] In some embodiments, the nucleic acid template is a mixture ofnucleic acid molecules, for instance a mixture of RNA molecules such asa preparation of total RNA, polyA RNA, or mRNA. In some embodiments, thestarting material contains ribosomal RNA, messenger RNA, transfer RNA ormixtures these. In some embodiments, the nucleic acid starting materialis DNA rather than RNA, and in yet other embodiments, it is a mixture ofboth DNA and RNA. In particular embodiments, polymerizing comprisespolymerizing a cDNA, for instance in a reverse transcription reactionwhere the template is an RNA molecule (or mixture thereof).

[0131] Also disclosed are methods wherein modified nucleic acids areincluded in random primers that are used to initiate polymerization of aprobe molecule, thereby introducing the modified nucleic acidsconsistently at the 5′ end of each probe molecule (such as cDNAs orfragments thereof). These methods maximize incorporation of modifiednucleic acids into the resulting probe, thereby providing enhancedsignal intensity and sensitivity in reactions using the probe, comparedto currently used methods.

[0132] In certain embodiments, the random primers include nucleotidesthat are modified by amine groups (such as aminoallyl moieties).Coupling of a fluorescent dye to the amine group can be performed aftersynthesis of the cDNA probe by reverse transcription. This novellabeling procedure provides for detection sensitivity at least two-foldenhanced compared to standard methods, and requires significantly lessstarting nucleic acid, be it DNA or RNA.

[0133] In other embodiments, the modified nucleotides comprise adetectable molecule, such as a fluorophore or hapten. In yet otherembodiments, the amplified nucleic acid template is labeled andcomprises a detectable molecule, such as a fluorophore or hapten.

[0134] In specific embodiments where the modified nucleotide in therandom primer comprises an amine-modified dNTP, the method furthercomprises coupling the modified nucleic acid probe to a label molecule(such as a fluorophore or hapten) to form a labeled probe (also referredto as a label-probe conjugate).

[0135] Also provided are modified random primers for use in thedisclosed methods. Specific examples of such primers are shown in SEQ IDNOs: 1-10, for instance specifically the primers referred to as P2 (SEQID NO: 1) or P4 (SEQ ID NO: 2).

[0136] Also provided are methods of producing a fluorescenthybridization probe, which includes contacting a template nucleic acidsample with a modified random primer comprising at least one aminoallyldNTP residue (such as an aminoallyl dUTP), polymerizing a nucleic acidmolecule complementary to a sequence in the template sample andincorporating one or more modified random primers into thatcomplementary molecule, to produce a modified complementary nucleotide.This modified complementary nucleotide can be contacted with anamine-reactive fluorescent label molecule, thereby producing afluorescent hybridization probe. In specific embodiments, the modifiedcomplementary nucleotide is contacted with an amine-reactive hapten, orother amine-reactive molecule or group. Also encompassed herein arehybridization probes produced using these methods.

[0137] In certain methods provided herein, aminoallyl dUTP (or anothermodified nucleotide) is included during a polymerizing step.

[0138] This disclosure also provides an improved method for randomprimer reverse transcription labeling of a nucleic acid hybridizationprobe. One provided improvement is the use of random primers modifiedwith at least one amine-substituted dNTP or fluorescent-dye modifieddNTP to prime (initiate) the reverse transcription reaction. Improvedhybridization probes produced by such methods are also provided.

[0139] Also provided are probe-labeling methods in which the templatemolecule is an amplified nucleic acid template. In one embodiment, theamplified template is RNA. In other embodiments, the amplified templateis DNA.

[0140] In certain disclosed methods the amplified template binds asecond primer under conditions sufficient to permit base-specifichybridization between the template and the second primer. The secondprimer can include a T3 promoter and a random 9-mer (T3N9; SEQ ID NO:12) and the second primer can be used in at least one round of cDNAsynthesis other than the first round. In particular embodiments, theT3N9 or a similar random primer is used for all steps of the method.

[0141] This disclosure also provides kits for producing a labeledhybridization probe or for probing an array, which kits include at leasta modified random primer.

[0142] IV. Modified Random Primers

[0143] DNA microarray technology has become one of the most importanttools for high throughput studies in medical research, with applicationsin the areas of gene discovery, gene expression, and genetic mapping.Much progress has been made for making high quality microarrays throughimproving the surface materials and fabrication techniques, but littlehas been achieved for the labeling methods to increase the detectionsignal and sensitivity which limits the application of DNA microarraytechnology in certain areas including clinical diagnosis. Geneexpression studies and clinical diagnosis using tissue biopsies or smallcell populations have in the past often been difficult due to thelimited availability of RNA, because prior methods of labeling cDNAprobes for microarray hybridization require substantial amounts of RNAto generate the probes.

[0144] Prior labeling techniques involve incorporating fluorescent dyeconjugated nucleotides such as Cy3-/Cy5- dUTP/dCTP, or other modifiednucleotides like aminoallyl dUTP (aa-dUTP), during probe polymerization(e.g., during reverse transcription of cDNA from mRNA). The optimalratio of dye-modified to unmodified nucleotide used is governed by twofactors: 1) that modified bases cause a deterioration in the strengthand specificity of binding of probes to their target DNAs, and 2) thatas many modified bases as possible have to be incorporated into probesto give good fluorescent signals. In practice, this trade-off limits theefficiency of probe labeling, and a large amount of starting RNA isrequired to produce labeled probe for each hybridization.

[0145] This disclosure provides methods for producing modified (e.g.,labeled) nucleic acid molecules useful as probes, for instance forhybridization to microarrays, which overcome disadvantages of priorlabeling methods. The probes provided herein have at least one label attheir 5′ end and they are more highly labeled than those produced usingprevious methods. The improved labeling is achieved through theincorporation of one or more chemically modified nucleotides (such asthose shown in FIG. 1) into random primers, which are then used toinitiate synthesis (polymerization) of the probe. These methods enablethe efficient production of probe nucleic acid incorporating themodification throughout the length of the molecule, withoutsubstantially decreasing label incorporation or efficiency of thepolymerization reaction.

[0146] Because nucleic acid probes produced using these methods areintensely labeled, less probe is needed in order to be reliablydetected, for instance in a hybridization reaction. As illustrated inthe Examples and accompanying figures, hybridization probes producedusing mono-modified primer embodiments (such as the primer referred toherein as P2) consistently provide reliable hybridization signals. Thus,the herein-described labeling methods can be used to reliably label verysmall amounts of starting material for analysis, such as expressionanalysis using microarrays.

[0147] One specific encompassed method is shown schematically in FIG. 2.In this illustrated method, mRNA (10) is used as the template to producemodified (in this case, fluorescently labeled) cDNA fragments (12). Amodified nucleotide (14) (such as the amine-modified nucleotideaminoallyl dUTP) is incorporated directly into random primers (16) thatare then used to prime reverse transcription (18) of the mRNA, producingamine-modified cDNA fragments (20). These fragments may be, but need notbe, full length cDNAs. After synthesis, label moieties (22) can be addedto the modified cDNA fragments (20) at the modification groups (14)(e.g., the amine groups of the amine-modified nucleotides) to producelabeled cDNA probe (12). This probe (12) in certain embodiments will bea mixture of labeled cDNA molecules, some of which will be fragments ofwhat would be considered “full-length” cDNAs.

[0148] Because modified nucleotides are incorporated directly into therandom primers, these methods result in reliable incorporation of a highlevel of reactive groups (and labels) into each probe molecule from asmall amount of starting total RNA, without substantially inhibiting theRT reaction. In some circumstances this provides a stronger fluorescentsignal, and may provide more consistent and reproducible fluorescentsignal, compared to standard RT methods using unlabeled random primersin the presence of modified individual nucleic acids. Thus, an effectiveprobe can be produced, and clear signals read from a microarray, evenwhen using significantly less starting nucleic acid (as little as about1-2 μg of total RNA). This enables microarray analysis of geneexpression from much smaller samples. This labeling protocol is verysimple and considerably less expensive than methods currently consideredto be state of the art.

[0149] In certain embodiments, particularly those comprising anamplification process, the amount of starting material (e.g., apreparation of nucleic acids, a lysed cell sample, etc.) may containless than about 1 μg of total RNA. In other embodiments, the amount ofstarting material may contain less than about 2 μg of total RNA, lessthan about 3 μg of total RNA, less than about 5 μg of total RNA, or lessthan about 10 μg of total RNA.

[0150] In some embodiments, particularly those comprising anamplification process, the starting material is DNA, for example genomicDNA. A sample used as the starting material may contain cellular genomicDNA, viral genomic DNA or both cellular and viral genomic DNA. In onespecific, non-limiting embodiment, the starting material is DNA obtainedfrom the body fluid of a subject infected with a virus. In anotherspecific, non-limiting embodiment, the starting material is DNA obtainedfrom a cell infected by a virus. This method can be used, for example,to amplify genomic DNA material from a subject infected with a virus orother types of pathogens. Labeled probes generated from amplified viralDNA, or from amplified DNA from another type of pathogen, can be used,for example with microarray detection, to detect the presence of a virusor pathogen in a subject. This detection method has a sensitivity andspecificity that is better than that of PCR (see Example 8, below).

[0151] In other embodiments, the starting material comprises as few asabout 1 cell, about 10 cells, about 100 cells, about 200 cells, about500 cells, about 750 cells, or about 1000 cells.

[0152] In one specific example, random hexamers and T7-oligo dT primersare used for the second and subsequent rounds of RNA amplification. Byway of further example, primers including the T3 promoter and a randomninemer (T3N9, SEQ ID NO: 12) can be used for the second and subsequentrounds of RNA amplification, thereby incorporating the T3 RNA polymerasepromoter sequence into the nucleic acid at random locations based on therandom ninemer (9-mer). Other promoters could be used.

[0153] Methods have also been developed and are described wherein a T3N9primer, or similar primer (e.g, with a longer or shorter random section)can be used at all steps in the amplification process. In one specific,non-limiting example, a T3N9 primer (SEQ ID NO: 12) is used to primecDNA synthesis from either an RNA or a DNA template. The T3 polymerasepromoter sequence is thus incorporated into the cDNA at the earlieststep in the synthesis of the random primers, at random locations basedon the random 9-mer. T3 DNA polymerase initiates RNA synthesis in one ormore rounds of RNA amplification. This method is simple, as it requiresonly a single primer throughout the procedure. In addition, it does notfavor the synthesis of 3′ products.

[0154] In some embodiments, additional amine-modified dUTP (or anotherdNTP) optionally can be included in the RT reaction, therebyincorporating additional amine-reactive groups into the cDNA duringsynthesis. This method can increase the labeling intensity and thereforeis suitable in certain circumstances.

[0155] Random primers have been widely used in labeling the DNA probeswith radioisotope from DNA template (Feinberg and Vogelstein, Anal.Biochem. 132:6-13, 1983; Swensen, BioTechniques, 20:486-491, 1996). Theyhave also been used in priming the cDNA synthesis from purified mRNA(Lear et al., BioTechniques 18:78-83, 1995; Allawi et al., RNA7:314-327, 2001). Because the largest portion in total RNA pool isribosomal RNA, using total RNA as template material to generate cDNAprobes may increase non-specific hybridization from microarrays.However, under highly stringent condition for hybridization and washingsteps, represented by the conditions presented herein, such problemshave been avoided.

[0156] Choice of Modification

[0157] Many examples of modified nucleotides are provided herein. Thechoice of which modification to use on a random primer provided hereinwill be influenced by the specific use to which the labeled probe is tobe put, and the detectable molecule to be coupled to the nucleotide. Forinstance, the detectable molecule must be able to couple with themodified nucleotide; one should comprise a nucleophilic reactive group,while the other contains an electron poor reactive center and a leavinggroup.

[0158]FIG. 1 and Table 1 show structures of specific examples ofmodified nucleotides and specific amine modified random primers (P2 andP4) made with these nucleotides. The amine-modified nucleotides areincorporated into the oligonucleotide primers during regular DNAchemical synthesis. Amine modified dT and dC are commercially availablein a form that can be used for DNA synthesis, for instance from Sigma(St. Louis, Mo.), or from Glen Research in Virginia. Additional modifiednucleotides, and sources, are listed in Example 6.

[0159] It is contemplated that other modified nucleotides are alsouseful in the described methods. For instance, nucleotides that carry alabel or other detectable molecule are considered to be modified, andcan be used to generate the modified primers employed in methodsdescribed herein. Methods for making such labeled nucleotides, andexamples thereof, are described in further detail in Example 6.

[0160] Synthesis of Oligonucleotide Primers

[0161] In vitro methods for the synthesis of oligonucleotides are wellknown to those of ordinary skill in the art; such conventional methodscan be used to produce primers for the disclosed methods. The mostcommon method for in vitro oligonucleotide synthesis is thephosphoramidite method, formulated by Letsinger and further developed byCaruthers (Caruthers et al., Chemical synthesis ofdeoxyoligonucleotides, in Methods Enzymol. 154:287-313, 1987). This is anon-aqueous, solid phase reaction carried out in a stepwise manner,wherein a single nucleotide (or modified nucleotide) is added to agrowing oligonucleotide. The individual nucleotides are added in theform of reactive 3′-phosphoramidite derivatives. See also, Gait (Ed.),Oligonucleotide Synthesis. A practical approach, IRL Press, 1984.

[0162] In general, the synthesis reactions proceed as follows: First, adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleosideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forinstances, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679,and 5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (e.g., the PolyPlex oligonucleotidesynthesizer from Gene Machines, San Carlos, Calif.). In addition, manycompanies will perform such synthesis (e.g., Sigma-Genosys, TX; OperonTechnologies, CA; Integrated DNA Technologies, IA; and TriLinkBioTechnologies, CA).

[0163] Modified nucleotides, such as aminoallyl dNTPs or dNTPs carryinga fluorescent dye (such as Cy3 or Cy5), can be incorporated into anoligonucleotide essentially as described above for non-modifiednucleotides.

[0164] Random primers may be generated using known chemical synthesisprocedures; randomness of the sequence may be introduced by providing amixture of nucleic acid residues in the reaction mixture at one or moreaddition steps (to produce a mixture of oligonucleotides with randomsequence). See, for instance, U.S. Pat. Nos. 5,043,272 and 5,106,727. Arandom primer preparation (which is a mixture of differentoligonucleotides, each of determinate sequence) can be generated bysequentially incorporating nucleic acid residues from a mixture of, forinstance, 25% of each of dATP, dCTP, dGTP, and dTTP, (or a modified dNTPsuch as aa-dUTP). Other ratios of dNTPs can be used (e.g., more or lessof any one dNTP, with the other proportions adapted so the whole amountis 100%). Likewise, in the synthesis of a random primer, the synthesizercan be programmed to introduce one or more known residues (such as oneor more specific nucleotide residues or modified nucleotide residues) ata defined location within the primer. For instance, a defined sequencecan be included at the 5′ or 3′ end of the primer, or in the middle ofthe primer (with random sequences to the 5′ and 3′ end), or acombination of these.

[0165] By way of further example, the following modified random primersare contemplated: TABLE 1 Amine Modified Random Primers Primer SequenceSEQ ID NO: P2 [AC6T]-NNNNNN * 1 P4 XXXXXN ** 2 Pr A [AC6T]-INNNNNN *** 3Pr B [AC6T]-[AC6T]-INNNNNN *** 4 Pr C [AC6T]-I-[AC6T]-INNNNNN *** 5 Pr D[AC6T]-II-[AC6T]-INNNNNN *** 6 Pr E [AC6T]-III-[AC6T]-INNNNNN *** 7 Pr F[AC6T]-IIII-[AC6T]-INNNNNN *** 8 Pr G [AC6T]-IIIII-[AC6T]-INNNNNN *** 9Pr H X-NNNNNN **** 10

[0166] Choice of Detectable Molecule

[0167] Though most of the examples presented herein refer to theaddition of a fluorescent label (particularly Cy3 or Cy5) to themodified nucleotide that is incorporated in a primer used in thedescribed methods, other detectable molecules are contemplated.

[0168] DNA molecules containing a primary amino group (e.g., attached tothe C6 or C2 carbon) can be coupled with a standard peptide or caninteract with any intermediate N-hydroxysuccinimide (NHS) ester. In anembodiment disclosed herein, amine modified dT and dC nucleotides areadded in place of thymidine and cytidine residues during oligonucleotidesynthesis. After deprotection of the modified group, the primary amineon (for instance) the C6 moiety is spatially separated from theoligonucleotide by a spacer arm with a total of 10 atoms, and can bereacted with a label molecule or attached to an enzyme or any otherreactive peptide or protein. Thus, for instance, the provided methodsfor making amine modified DNA can be used to produce modified probemolecules that comprise a peptide antigen or single chain antibody,which can be used in detection assays involving antigen and antibodyreactions.

[0169] Thus, in particular embodiments, the provided primers are linkedto a hapten such as biotin, or a fluorescent dye. For instance, anyNHS-ester dyes can be used in DNA probe labeling with the provided aminemodified random primers.

[0170] In addition, it is contemplated that in those embodiments inwhich the modification of the nucleotide is a label (e.g., a fluorescentdye molecule) or other detectable molecule, the modified primer is thelabeled primer and can be used to produce labeled probe withoutrequiring a subsequent chemical modification.

[0171] Applications

[0172] Because the disclosed labeling methods require very littlestarting material, even as little as one cell, these methods open upconventional cDNA microarray analysis to entire new fields of research,particularly those in which the source material was heretofore tooscarce to permit cDNA array analysis (e.g., for samples acquired by fineneedle aspirates or micro-dissection, or experimental models studyingembryonic tissue or small organisms). For instance, these methods can beused to study specific cell populations within the brain or fromembryonic cell samples (e.g., to study embryonic development). Geneexpression within individual white blood cells, such as those fromperipheral blood cells, or other potentially unique cells, can beassessed using these methods. Within a tissue biopsy, different cellpopulations can be sampled (e.g., through laser capture microdissection)and the expression levels of genes in the different cell populationsassayed.

[0173] Similarly to regular random primers, the provided amine modifiedrandom primers also can be used in many applications such as RT-PCR,FISH and others in which fluorescent dyes are utilized for signaldetection. For instance, the provided modified primer labeling systemcan be used to make labeled probes from DNA templates using E. coli DNApolymerase I by random priming labeling.

[0174] Previous Methods of Labeling cDNA Probes

[0175] For the sake of comparison, the following is a representativeexample of prior methods for labeling hybridization cDNA probes for usein microarray analysis. This method produces fluorescently labeled cDNAusing traditional primers (oligo(dT) or unmodified random primers) andreverse transcription PCR. The presented method is adapted from thoseavailable at the Internet site of the National Human Genome ResearchInstitute, National Institutes of Health, Bethesda, Md.

[0176] Using an anchored oligo dT primer, the primer is annealed to theRNA in the following 17 μl reaction (use a 0.2 ml PCR tube so thatincubations can be carried out in a PCR cycler): Component for Cy5labeling for Cy3 labeling Total RNA (>7 mg/ml) 150-200 μg 50-80 μgAnchored primer (2 μg/μl)    1 μl    1 μl DEPC H₂O to 17 μl to 17 μl

[0177] If using an oligo dT(12-18) primer, the primer is annealed to theRNA in the following 17 μl reaction: Component for Cy5 labeling for Cy3labeling Total RNA (>7 mg/ml) 150-200 μg 50-80 μg dT(12-18) primer (1μg/μl)    1 μl    1 μl DEPC H₂O to 17 μl to 17 μl

[0178] The incorporation rate for Cy5-dUTP is less than that ofCy3-dUTP, so more RNA is labeled to achieve more equivalent signal fromeach labeled species.

[0179] The samples are then heated to 65° C. for 10 minutes and cooledon ice for 2 minutes. To each tube, add 23 μl of reaction mixture(below) containing either Cy5-dUTP or Cy3-dUTP nucleotide, mix well bypipetting and use a brief centrifuge spin to concentrate the reaction inthe bottom of the tube. Reaction Mixture μl 5x first strand buffer 8 10xlow T dNTPs mix 4 Cy5 dUTP or Cy3 dUTP (1 mM) 4 0.1 M DTT 4 RNasin (30u/μl) 1 Superscript II (200 u/μl) 2 Total volume 23

[0180] Superscript polymerase is sensitive to denaturation at air/liquidinterfaces, so care is exercised to suppress foaming in handling of thisreaction.

[0181] The polymerization reaction is incubated at 42° C. for 30minutes. An additional 2 μl of Superscript II is added, well mixed intothe reaction volume, and incubated at 42° C. for an additional 30-60minutes.

[0182] To stop the reaction, 5 μl of 0.5M EDTA is added, followed by 10μl 1N NaOH. The sample is incubated at 65° C. for 30-60 minutes tohydrolyze residual RNA, then cooled to room temperature. The reactionmust be stopped by addition of EDTA before the NaOH is added, sincenucleic acids precipitate in alkaline magnesium solutions. Also, thepurity of the sodium hydroxide solution is important; slightcontamination or long term storage in a glass vessel can produce asolution that will degrade the Cy5 dye, turning the solution yellow.

[0183] The reaction is neutralized by adding 25 μl of 1M Tris-HCl (pH7.5). The labeled cDNA is desalted using a MicroCon 100 cartridge. Theneutralized reaction, 400 μl of TE pH 7.5 and 20 μg of human C0t-1 DNAare added to the cartridge and mixed by pipetting. The column is spunfor 10 minutes at 500×g, then washed by adding 200 μl TE pH 7.5. Thesample is concentrated to about 20-30 μl by spinning at 500×g forapproximately 8-10 minutes.

[0184] Alternatively, a smaller pore MicroCon 30 cartridge can be usedto speed the concentration step. In this case, centrifuge the first washis performed for approximately 4.5 minutes at 16,000×g and the second(200 μl wash) for about 2.5 minutes at 16,000×g.

[0185] The neutralized and desalted sample is recovered by inverting theconcentrator cartridge over a clean collection tube and spinning forthree minutes at 500×g.

[0186] In some cases, the Cy5-labeled cDNA will form a gelatinous blueprecipitate that is recovered in the concentrated volume. This indicatesthat the sample was contaminated. The more extreme the contamination,the greater the fraction of cDNA the will be captured in this gel. Evenif heat solubilized, this material tends to produce uniform,non-specific binding to the DNA targets.

[0187] When concentrating by centrifugal filtration, the times requiredto achieve the desired final volume are variable. Overly long spins canremove nearly all the water from the solution being filtered. Whenfluor-tagged nucleic acids are concentrated on the filter in thisfashion, they are very hard to remove from the cartridge. It isbeneficial to approach the desired volume by conservative approximationsof the required spin times. If control of volumes proves difficult, thefinal concentration can be achieved by evaporating liquid in thespeed-vacuum. Vacuum evaporation, if not to complete dryness, does notdegrade the performance of the labeled cDNA.

[0188] A 2-3 μl aliquot of the Cy5 labeled cDNA probe can be used forquality analysis (leaving 18-28 μl for hybridization). Run this probe ona 2% agarose gel (for instance, 6 cm wide×8.5 cm long, 2 mm wide teeth)in Tris Acetate Electrophoresis Buffer (TAE). For maximal sensitivitywhen running samples on a gel for fluor analysis, loading buffer withminimal dye is used, and ethidium bromide is not added to the gel orrunning buffer.

[0189] The resultant gel can be scanned on a Molecular Dynamics Stormfluorescence scanner (setting: red fluorescence, 200 micron resolution,1000 volts on PMT). Successful labeling produces a dense smear of probefrom 400 bp to>1000 bp, with little pile-up of low molecular weighttranscripts. Weak labeling and significant levels of low molecularweight material indicate a poor labeling reaction. A fraction of theobserved low molecular weight material is unincorporated fluornucleotide, and should be expected in any reaction.

[0190] Computer Assisted (Automated) Detection and Analysis of ArrayHybridization

[0191] The data generated by assaying an array can be analyzed usingknown computerized systems. For instance, the array can be read by acomputerized “reader” or scanner and quantification of the binding ofprobe to individual addresses on the array carried out using computeralgorithms. Likewise, where a control probe (such as a probe preparedfrom a control cell or sample with known expression levels) has beenused, computer algorithms can be used to normalize the hybridizationsignals in the different spots of the array. Such analyses of an arraycan be referred to as “automated detection” in that the data is beinggathered by an automated reader system.

[0192] In the case of labels that emit detectable electromagnetic waveor particles, the emitted light (e.g., fluorescence or luminescence) orradioactivity can be detected by very sensitive cameras, confocalscanners, image analysis devices, radioactive film or a Phosphoimager,which capture the signals (such as a color image) from the array. Acomputer with image analysis software detects this image, and analyzesthe intensity of the signal for each probe location in the array.Signals can be compared between spots on a single array, or betweenarrays (such as a single array that is sequentially probed with multipledifferent probe molecules), or between the labels of different probes ona single array.

[0193] Computer algorithms can also be used for comparison between spotson a single array or on multiple arrays. In addition, the data from anarray can be stored in a computer readable form.

[0194] Certain examples of automated array readers (scanners) will becontrolled by a computer and software programmed to direct theindividual components of the reader (e.g., mechanical components such asmotors, analysis components such as signal interpretation and backgroundsubtraction). Optionally software may also be provided to control agraphic user interface and one or more systems for sorting,categorizing, storing, analyzing, or otherwise processing the dataoutput of the reader.

[0195] To “read” an array, an array that has been assayed with adetectable probe to produce binding (e.g., a binding pattern) can beplaced into (or onto, or below, etc., depending on the location of thedetector system) the reader and a detectable signal indicative of probebinding (hybridization) detected by the reader. Those addresses at whichthe probe has bound to an immobilized nucleic acid on the array providea detectable signal, e.g., in the form of electromagnetic radiation.These detectable signals could be associated with an address identifiersignal, identifying the site of the “positive” hybridized spot. Thereader gathers information from the addresses, associates it with theaddress identifier signal, and recognizes addresses with a detectablesignal as distinct from those not producing such a signal. Certainreaders are also capable of detecting intermediate levels of signal,between no signal at all and a high signal, such that quantification ofsignals at individual addresses is enabled.

[0196] Certain readers that can be used to collect data from the arrays,especially those that have been probed using a fluorescently taggedprobe, will include a light source for optical radiation emission. Thewavelength of the excitation light will usually be in the UV or visiblerange, but in some situations may be extended into the infra-red range.A beam splitter can direct the reader-emitted excitation beam into theobject lens, which for instance may be mounted such that it can move inthe x, y and z directions in relation to the surface of the arraysubstrate. The objective lens focuses the excitation light onto thearray, and more particularly onto the (polypeptide) targets on thearray. Light at longer wavelengths than the excitation light is emittedfrom addresses on the array that contain fluorescently-labeled probemolecules (i.e., those addresses containing a nucleic acid moleculewithin a spot containing a nucleic acid molecule to which the probebinds).

[0197] In certain embodiments, the array may be movably disposed withinthe reader as it is being read, such that the array itself moves (forinstance, rotates) while the reader detects information from eachaddress. Alternatively, the array may be stationary within the readerwhile the reader detection system moves across or above or around thearray to detect information from the addresses of the array. Specificmovable-format array readers are known and described, for instance inU.S. Pat. No. 5,922,617, hereby incorporated in its entirety byreference. Examples of methods for generating optical data storagefocusing and tracking signals are also known (see, for example, U.S.Pat. No. 5,461,599, hereby incorporated in its entirety by reference).

[0198] For the electronics and computer control, a detector (e.g., aphotomultiplier tube, avalanche detector, Si diode, or other detectorhaving a high quantum efficiency and low noise) converts the opticalradiation into an electronic signal. An op-amp first amplifies thedetected signal and then an analog-to-digital converter digitizes thesignal into binary numbers, which are then collected by a computer.

[0199] The following examples are provided to illustrate certainparticular features and/or embodiments. These examples should not beconstrued to limit the invention to the particular features orembodiments described.

EXAMPLES Example 1 Production of Primers for cDNA Labeling

[0200] Oligo (dT) 12-18 primer (referred to herein as P0) was purchasedfrom GIBCO BRL Life Technologies (Rockville, Md.); it is supplied in apre-made solution at a concentration of 500 μg/ml.

[0201] Unmodified, random hexamer primer (referred to herein as P1) waspurchased from Operon Technologies (New Orleans, La.) and was dissolvedin DEPC treated H₂O at the concentration of 1 μg/μl.

[0202] Currently, amine-modified nucleotides dT and dC are availablefrom Glen Research (Sterling, Va.). FIG. 1 shows the structures of theseamine-modified nucleotides. Using these modified nucleotides, twodifferent amine modified random primers (referred to herein as P2 andP4; Table 2) were synthesized using in vitro chemical synthesis usingthe phosphoramidite method (Caruthers et al., Chemical synthesis ofdeoxyoligonucleotides, in Methods Enzymol. 154:287-313, 1987). Theoligonucleotides were dissolved in DEPC treated H₂O at the concentrationof 1 μg/μl for use in reverse transcription reactions. TABLE 2 AmineModified Random Primers Primer Sequence (5′ to 3′ ) Manufacturer SEQ IDNO: P2 [AC6T]NNNNNN * SIGMA Genosys (The 1 Woodland, TX) P4 XXXXXN **TriLink BioTechnologies 2 (San Diego, CA)

Example 2 Generation of Fluorescent Probe

[0203] This example describes methods for producing labeled cDNA usingamine modified primers P2 and P4 and reverse transcription in thepresence of aminoallyl dUTP.

[0204] Template RNA

[0205] RNAs from mouse C2 and NIH 3T3 cell lines were isolated usingTRIzol reagent from GIBCO BRL Life Technologies (Rockville, Md.)followed by extraction with RNeasy kit from Qiagen (Valencia, Calif.).RNAs from mouse 18-day embryo and liver were also extracted with thecombination of TRIzol reagent and RNeasy kit.

[0206] Production of cDNA Probe

[0207] Primer (P0, P1, P2, or P4) was annealed to the RNA in thefollowing manner:

[0208] Primer (2 μl), total RNA (0.1-5 μg in 15.5 μl), and RNaseinhibitor (1 μl, Promega, Madison, Wis.) were mixed, and the RNA/primermixture incubated at 70° C. for 10 minutes, then chilled on iceimmediately for 10 minutes to encourage annealing of the primers.

[0209] A 17 μl aliquot of the RT mix (6 μl 5×first strand buffer(provided with SSII RT), 6 μl 5×aa-dUTP/dNTPs, 3 μl 0.1 M DTT, and 2 μlSuperScript II Reverse transcriptase (SSII RT; GIBCO BRL LifeTechnologies, Rockville, Md.) was added to each primer-RNA mix, for atotal volume of 30 μl, and the sample incubated at 42° C. for 2 hours topermit reverse transcription of the cDNA. aa-dUTP(5-[3-aminoallyl]-2′-deoxyuridine 5′-triphosphate) was from Sigma, St.Louis, Mo.

[0210] The reverse transcription reaction was stopped by the addition of10 μl of 0.5M EDTA. RNA was degraded from the reaction mixture by adding10 μl of 1N NaOH, and incubating the sample at 65° C. for 30 minutes.The reaction was neutralized by adding 10 μl of 1M HCl.

[0211] Various methods can be used to clean up the neutralized cDNAsample. In one method, the cDNA was cleaned up using a MinElute PCRpurification kit. The microcentrifuge tube was filled with 300 μl BufferPB and 60 μl of the neutralized reaction solution, essentially asprovided by the manufacturer, was added to the Buffer PB. The MinElutecolumn was placed in a 2 ml collection tube in a suitable rack. To bindDNA, the sample was applied to the MinElute column and centrifuged for 1minute. For optimal recovery, all traces of sample were transferred tothe column. The flow-through was poured back into the column andcentrifuged again for 1 minute. The flow-through was then discarded. TheMinElute column was washed by placing it back into the originalcollection tube, adding 750 μl of Buffer PE then incubating it for 5minutes at room temperature. The column was then centrifuged for 1minute, the flow-through discarded and the MinElute column placed backin the same tube. The column was centrifuged for an additional 1 minuteat maximum speed to remove residual buffer PE, then placed in a clean1.5 ml microcentrifuge tube.

[0212] To elute the DNA, 10 μl of water (pH between 7.0 and 8.5) wasadded to the center of the membrane within the column. After 1 minutethe column was centrifuged for 5 minutes. The average eluate was 9 μlout of the 10 μl applied. DNA was eluted from the column twice more with10 μl of water, collecting a total of 27 μl of purified cDNA.

[0213] In another method, the neutralized cDNA sample was cleaned upusing a MicroCon 100 concentrator cartridge (Millipore, Bedford, Mass.).The cartridge was primed with 450 μl of ddH₂O, then the cartridge wasspun at 13,000 rpm for about 3 minutes. The flow-through was discarded,and the cDNA on the cartridge was washed twice with 500 μl of ddH₂O. ThecDNA sample was eluted from the cartridge, and dried in a speed vacuum.

[0214] Coupling

[0215] Various coupling methods can be used. Monofunctional NHS-esterCy3 and Cy5 dyes and the dNTPs used in the coupling were from AmershamPharmacia (Piscataway, N.J.).

[0216] In one method, 3 μl of 1M sodium bicarbonate, pH 9.3 was added tothe 27 μl of cDNA purified from the MinElute column, followed by theaddition of 1 μl of dye solution (NHS-ester Cy3 or Cy5, 62.5 μg/μl inDMSO). The solution was mixed by pipetting up and down several times.The tubes were wrapped in aluminum foil, and incubated at roomtemperature for one hour in an orbital shaker (USA Scientific, Ocala,Fla.)).

[0217] In another method, a 5×aa-dUTP/dNTPs solution was made asfollows: 10 μl each of dATP (100 mM), dGTP (100 mM), and dCTP (100 mM);4 μl of aa-dUTP (100 mM), 6 μl of dTTP (100 mM) were combined with 360μl of DEPC-treated H₂O. The monofunctional NHS-ester Cy3 and Cy5 dyeswere first resuspended in 72 μl of dd H₂O and aliquots of 4.5 μl wereplaced in tubes. The aliquots were dried in a speed vacuum and stored in−20° C. freezer. The dried dyes were re-suspended in 4.5 μl of 100 mMsodium bicarbonate before mixing with cDNA made from reversetranscription reactions.

[0218] The cDNA sample dried in a speed vacuum was resuspended in 4.5 μlwater. Pre-dried aliquots of monofunctional NHS-ester Cy3 or Cy5 dyewere resuspended in 4.5 μl of 100 mM NaBicarbonate Buffer (pH 9.0). Onealiquot of cDNA was mixed with one aliquot of Cy3 or Cy5, and thesamples incubated at RT for 1 hour in dark to couple the fluorescent dyeto the modified cDNA.

[0219] Quenching and Cleanup

[0220] The fluorescence was quenched by adding 4.5 μl of 4Mhydroxylamine to the dye-coupled cDNA, and incubating the mixture at RTfor 30 minutes in dark. The labeled sample was cleaned up using a QiagenQia-quick PCR purification kit (Qiagen, Valencia, Calif.) as follows:

[0221] The Cy3 and Cy5 labeled reactions were mixed, and 30 μl water and500 μl Buffer PB (provided with the kit) added. This diluted sample wasapplied to the Qia-quick column, and the column spun at 13,000 rpm infor 1 minute. The flow-through was removed by aspiration, and the columnwashed twice with 750 μl Buffer PE (provided with the kit), spun out for1 minute and the flow-through aspirated off each time. The column wasthen transferred to a fresh tube, and 30 μl of Buffer EB (provided withthe kit) added to column. This was incubated for 1 minute at RT to elutethe probe, then the column spun at 13,000 rpm for 1 minute and theeluate collect. The elution step was repeated, the sample combined withthe first eluate, for a total collected volume of approximately 60 μl.

Example 3 Microarray Hybridization

[0222] This example provides a method for analyzing cDNA microarraysusing labeled probe produced by the amine modified random primer method,such as that produced by the method of Example 2. The signal generatedfrom microarray hybridization using cDNAs produced using amine-modifiedrandom primers is more consistent and more reliable than that obtainedwith previously known methods that use traditional random or oligo-dTprimers.

[0223] Microarray

[0224] cDNA microarrays with 10,752 mouse clones were fabricated onglass slides using OmniGrid from GeneMachines (San Carlos, Calif.) usingstandard techniques.

[0225] Hybridization and Analysis

[0226] The labeled cDNA eluate produced in Example 2 was dried in aspeed vacuum, and brought up in ddH₂O to a final volume of 23 μl. Tothis was added 4.5 μl of 20×SSC, 2 μl of poly A (10 mg/ml), and 0.6 μlof 10% SDS. The nucleic acids were denatured at 100° C. for 2 minutes,then applied to a prepared microarray. The microarray was permitted tohybridize by incubation for 16-24 hours in a 65° C. water bath.

[0227] After hybridization, the slide was washed at room temperature for10 minutes each in the following solutions: (a) 0.5×SSC, 0.01% SDS, (b)0.06×SSC. The washed slide was spun (in a tube or a slide rack) at 800rpm at room temperature for five minutes to dry.

[0228] Microarray hybridization images were scanned with GenePix 4000Ascanner from Axon (Foster City, Calif.), and the resultant data analyzedwith IPLab (Fairfax, Va.) and ArraySuite (Chen, NHGRI). To determine thereliability of each ratio measurement, a set of quality indicators wasused. An intensity measurement in either channel is determined to beunreliable if it fails to satisfy any one of the followingconditions: 1) The number of pixels associated with the element must besufficiently large. 2) The local background must be flat. 3) The signalconsistency within the target area must be uniform. 4) The majority ofthe signal pixels should not be saturated. For each ratio measurement,red (R)/green (G), one further condition is imposed-the average signal,(R+G)/2, must be three times the noise level (Chen et al. BiomedicalOptics. 2, 364-374, 1997).

RESULTS AND DISCUSSION

[0229] P0 Versus P2, Using Half as Much Template RNA

[0230] Oligo dT primer (P0) and amine modified random primer (P2) weredirectly compared. 2.5 μg total RNA was used for labeling with randomprimer P2; twice that amount was used with oligo dT primer P0. The twolabeled probes were then hybridized to each of two identical arrays onthe same slide. The slide was scanned at same laser power and PMT level.The images were processed and analyzed with IPLab/ArraySuite. Thehybridization color pattern with the amine modified random primer P2 wasexactly same as the pattern with the oligo dT primer. While the amountof RNA used with the amine modified random primer P2 was only half thatused with oligo dT primer P0, the observed hybridization intensitieswere similar to those obtained with the P2 primer.

[0231] The Pearson's correlation was calculated from the two ratio setsand scatter plots were generated; the calculated Pearson's r value was0.8006 for the P0/P2 comparison. This was similar to that observed whentwo arrays both hybridized with probe prepared with primer P2 werecompared (Pearson's r value of 0.8143).

[0232] P0 Versus P2, Using 10-fold Less Template RNA

[0233] Microarray hybridization was compared using probes produced withamine modified random primer P2 (Y-axis) and direct labeling techniqueusing oligo dT primer P0 (X-axis). Five μg of either mouse NIH 3T3 ormouse C2 total RNA was used to produce labeled cDNA with amine modifiedrandom primer P2; in contrast, 50 μg of mouse NIH 3T3 or C2 total RNAwas used to obtain a readable signal using the traditional directlabeling protocol primed by the oligo(dT) primer P0. By using the aminemodified random primer P2, it was demonstrated that only one tenth ofstarting material was needed to generate very similar hybridizationsignal intensities.

[0234] Differentially Expressed Genes (P0 Versus P2, Using 10-fold LessTemplate RNA)

[0235] Table 3 includes a list of 95 genes that are differentiallyexpressed in mouse 3T3 versus C2 cells (3T3/C2 ratios≧3 or ≦⅓). Thetable shows the results of six array experiments. Three 9568-elementarrays were interrogated with oligo-dT primed probes, and three otherswere interrogated with amine modified hexamer primed probes. Fifty pg ofRNA were used for each oligo-dT primed labeling and 5 μg were used foreach modified hexamer primed labeling. Array images were analyzed usingArraySuite software. Low quality ratios were filtered as describedabove.

[0236] When genes were searched that were 3-fold over-or under-expressedby the two cell lines studied, 99 genes were found with the oligo-dTpriming method and 102 genes were found with the amine-modified method.Among these, 95 genes were the same. The ratios of the 95 genes thatwere differentially expressed are quite consistent among all sixexperiments. Elements representing the RhoB and four-and-a-halfLIMdomains 1 transcripts were printed two and three times on the array,respectively. These genes appear to be expressed at a higher level in C2than 3T3 cells, and it is convincing that all elements representing themshowed similar ratios. The four-and-a-half LIM domains protein is knownto be produced in cardiac and skeletal muscle. TABLE 3 GenBank NoUnigene No dTa dTb dTc P2a P2b P2c Clone description AI849214 Mm.10533018.3218 17.5265 16.2183 20.7580 25.5790 22.7692 whey acidic proteinAI848293 Mm.34507 6.9711 5.3423 3.5613 8.1450 6.4472 6.7642 ESTsAI847098 Mm.29982 5.2438 4.5541 4.5709 5.6932 5.1469 5.7707 ERO1-like(S. cerevisiae) AI852317 Mm.4063 3.7839 3.5895 4.4260 5.4368 5.08305.2313 N-myc downstream regulated 1 AI844828 Mm.2834 3.7150 3.71053.9967 5.1164 4.9883 4.7269 glycine transporter 1 AI846827 Mm.706675.2250 4.0641 3.4577 4.6474 4.2576 4.2443 Mus musculus, Similar tooxidation resistance 1 AI843085 Mm.157648 5.5280 4.4538 4.7526 4.51774.0203 4.3967 RIKEN cDNA 5730403B10 gene AI842716 Mm.140158 5.50155.8588 5.1314 4.4732 4.4336 4.4295 cytochrome P450, 51 AI836864 Mm.47046.6261 5.7066 3.7317 4.4534 3.9560 4.4178 forkhead box G1 AI853347Mm.21884 4.0523 3.9847 3.3449 4.4364 4.7968 4.3672 ESTs, Weakly similarto GTPase-activating protein SPA-1 AI843677 Mm.45357 3.7376 3.59433.5423 3.8309 3.4541 3.3920 Erbb2 interacting protein AI838612 Mm.146013.0926 3.3974 3.2623 3.6027 3.4499 3.4159 glutathione S- transferase, mu2 AI848205 Mm.35844 3.6669 3.3875 3.1423 3.4911 3.0100 3.1019 growtharrest specific 5 AI850589 Mm.22627 3.7784 3.1037 3.1616 3.2339 3.24073.6818 epidermal growth factor receptor pathway substrate 15 AI852765Mm.24193 0.3300 0.3343 0.3183 0.3254 0.2847 0.3249 glypican 1 AI836264Mm.4871 0.1492 0.1253 0.1183 0.3200 0.3100 0.2357 tissue inhibitor ofmetalloproteinase 3 AI844851 Mm.10406 0.3209 0.3235 0.2910 0.3243 0.29930.3025 RIKEN cDNA 3110001M13 gene AI851985 Mm.29586 0.2668 0.2559 0.22780.3233 0.2814 0.3107 RIKEN cDNA 2610024P12 gene AI845475 Mm.30811 0.10310.1333 0.1210 0.3180 0.3200 0.3100 ESTs AI853172 Mm.27173 0.2968 0.31330.3032 0.3132 0.2847 0.3100 ectoplacental cone sequence AI835858Mm.27685 0.2834 0.2925 0.2512 0.3114 0.3067 0.2751 ESTs, Highly similarto tropomyosin 4 [Rattus norvegicus] AI836045 Mm.29976 0.2461 0.32020.2812 0.3016 0.3236 0.2702 septin 5 AI843823 Mm.7414 0.1481 0.16900.1445 0.2971 0.3129 0.2507 neuron specific gene family member 1AI844342 Mm.182255 0.1773 0.2039 0.2446 0.2833 0.3164 0.3083 CD97antigen AI835331 Mm.544 0.2802 0.3336 0.3057 0.2829 0.1995 0.2646phosphoprotein enriched in astrocytes 15 AI845602 Mm.4146 0.2438 0.26680.3188 0.2727 0.2349 0.2469 platelet derived growth factor receptor,beta polypeptide AI838302 Mm.4426 0.2816 0.2966 0.3223 0.2702 0.24660.2872 Cd63 antigen AI835546 Mm.3117 0.2023 0.2238 0.2903 0.2696 0.30220.3240 T-cell death associated gene AI853531 Mm.21679 0.2340 0.30060.3272 0.2691 0.2573 0.2708 RIKEN cDNA 1300002F13 gene AI842302 Mm.41390.3176 0.3029 0.3261 0.2652 0.2259 0.2783 rhotekin AI835620 No Data0.2793 0.3169 0.3180 0.2637 0.2298 0.2679 No Data AI845774 Mm.856 0.27990.2757 0.3172 0.2630 0.2362 0.2575 transmembrane 4 superfamily member 1AI838659 Mm.262 0.2496 0.2866 0.3001 0.2484 0.2192 0.2592 ras homologgene family, member C AI848618 Mm.29010 0.1939 0.2150 0.2075 0.24730.2205 0.2216 membrane bound C2 domain containing protein AI851997Mm.29010 0.2759 0.2851 0.3298 0.2462 0.2379 0.2648 membrane bound C2domain containing protein AI852812 Mm.2308 0.2209 0.2669 0.3063 0.24090.2236 0.2485 hemoglobin Z, beta- like embryonic chain AI844356 Mm.10170.2547 0.2658 0.2582 0.2261 0.2191 0.2255 esterase 10 AI851647 Mm.222400.2365 0.2571 0.2440 0.2219 0.2185 0.2236 ESTs, Weakly similar to SH3BGRprotein AI838551 Mm.2792 0.1605 0.1832 0.1807 0.2191 0.1398 0.2238prostaglandin- endoperoxide synthase 1 AI842654 Mm.8180 0.2336 0.25950.2941 0.2182 0.2249 0.2627 lymphocyte antigen 6 complex AI841122Mm.39804 0.2427 0.2581 0.3048 0.2139 0.2408 0.2015 EST AI838653Mm.181074 0.2615 0.2885 0.3198 0.2073 0.2179 0.2407 RIKEN cDNA2610001E17 gene AI838959 Mm.16537 0.1483 0.1504 0.2370 0.2014 0.29430.2463 actin, alpha 2, smooth muscle, aorta AI842847 Mm.8245 0.20130.2803 0.2512 0.1975 0.1770 0.1926 tissue inhibitor of metalloproteinaseAI838351 No Data 0.1422 0.1998 0.0999 0.1913 0.3317 0.2076 No DataAI837390 Mm.43278 0.1418 0.1444 0.1499 0.1882 0.2873 0.2535 olfactomedinrelated ER localized protein AI844326 Mm.194675 0.2317 0.2675 0.22900.1847 0.0958 0.1462 EST AI839057 No Data 0.2107 0.2988 0.2685 0.18060.2179 0.2184 No Data AI838085 Mm.687 0.1668 0.1773 0.2450 0.1781 0.22980.2301 aplysia ras-related homolog B (RhoB) AI837494 Mm.39836 0.16040.1709 0.2824 0.1768 0.1658 0.1247 ESTs, Weakly similar to T14318ubiquitin- protein ligase E3- alpha AI836532 Mm.196484 0.1481 0.14640.1405 0.1645 0.1642 0.1756 EST AA408841 AI835609 Mm.1956 0.0364 0.07760.0791 0.1608 0.2416 0.1599 neurofilament, light polypeptide AI842984Mm.980 0.1258 0.1350 0.1376 0.1602 0.2456 0.1732 tenascin C AI849378Mm.2769 0.1639 0.1670 0.1944 0.1545 0.1712 0.2004 MARCKS-like proteinAI839275 Mm.738 0.1356 0.1868 0.2704 0.1503 0.2651 0.1883 procollagen,type IV, alpha 1 AI844626 Mm.29975 0.0684 0.1024 0.1284 0.1489 0.19560.1716 RIKEN cDNA 1810003P21 gene AI835201 Mm.8739 0.1115 0.1536 0.14020.1454 0.1709 0.1867 sarcoglycan, epsilon AI844312 Mm.3091 0.1443 0.24000.2183 0.1432 0.2094 0.1778 epsin 1 AI841755 Mm.687 0.1340 0.1510 0.13450.1427 0.1610 0.1485 aplysia ras-related homolog B (RhoB) AI838813Mm.192516 0.1338 0.1664 0.1652 0.1416 0.1249 0.1655 EST AI839735Mm.37751 0.1409 0.1558 0.1463 0.1403 0.1138 0.1486 ESTs AI837031Mm.157662 0.0520 0.0994 0.1407 0.1260 0.0776 0.0931 synaptotagmin 13AI840673 Mm.29924 0.0846 0.0945 0.1128 0.1237 0.1111 0.1437ADP-ribosylation-like factor 6 interacting protein AI841538 Mm.410090.1166 0.1329 0.2839 0.1210 0.1168 0.1004 Nedd4 WW-binding protein 4AI847958 Mm.20246 0.1447 0.1526 0.2049 0.1173 0.0934 0.1017 RIKEN cDNA2410004D18 gene AI840633 Mm.38021 0.0477 0.1194 0.1215 0.1122 0.08230.0391 carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 AI843323Mm.3900 0.1334 0.1957 0.2642 0.1120 0.0902 0.1358 latent transforminggrowth factor beta binding protein 2 AI849869 Mm.34113 0.1241 0.13360.1955 0.1120 0.1015 0.1198 VPS10 domain receptor protein SORCS 2AI840335 Mm.39154 0.0928 0.1347 0.1007 0.1104 0.1833 0.1133 EST AI840972Mm.29580 0.2618 0.3083 0.3024 0.1059 0.1794 0.1681 superiorcervicalganglia, neural specific 10 AI847162 Mm.29357 0.0973 0.0696 0.20180.1050 0.1264 0.1312 RIKEN cDNA 1300017C10 gene AI843174 Mm.29924 0.12840.1426 0.1479 0.1044 0.1134 0.1473 ADP-ribosylation-like factor 6interacting protein AI839366 Mm.28947 0.0651 0.1159 0.1742 0.1021 0.12780.1395 ESTs AI840692 No Data 0.1394 0.1457 0.1741 0.0917 0.1456 0.1644No Data AI835703 Mm.29975 0.0961 0.0827 0.0714 0.0868 0.1302 0.1381RIKEN cDNA 1810003P21 gene AI836865 Mm.44102 0.0503 0.0643 0.1129 0.08420.1727 0.1572 ESTs AI842983 Mm.192586 0.0702 0.1325 0.1346 0.0785 0.15550.1091 EST AI839950 Mm.3126 0.0492 0.0610 0.0989 0.0781 0.2076 0.1304four and a half LIM domains 1 AI844604 Mm.3126 0.1263 0.1328 0.14650.0750 0.0188 0.0613 four and a half LIM domains 1 AI836826 Mm.29760.0747 0.0764 0.0755 0.0747 0.0918 0.0759 glycoprotein 38 AI850497Mm.41072 0.1133 0.1862 0.2509 0.0743 0.1009 0.0891 ESTs, Highly similarto LOX5 mouse arachidonate 5- lipoxygenase AI835403 Mm.142729 0.09650.1012 0.1014 0.0620 0.0778 0.0579 thymosin, beta 4, X chromosomeAI848096 Mm.17951 0.1483 0.1711 0.1888 0.0580 0.1324 0.1233 erythrocyteprotein band 4.1-like 3 AI843282 Mm.181021 0.0955 0.1120 0.1453 0.05290.0995 0.1095 procollagen, type IV, alpha 2 AI842554 Mm.192583 0.05770.0889 0.0962 0.0428 0.1088 0.0815 ESTs AI842681 Mm.20904 0.0702 0.04880.1056 0.0405 0.0375 0.0487 cartilage associated protein AI835976Mm.17951 0.0491 0.0372 0.0362 0.0392 0.0591 0.0431 erythrocyte proteinband 4.1-like 3 AI836468 Mm.30059 0.0495 0.0491 0.1289 0.0345 0.06900.0530 myristoylated alanine rich protein kinase C substrate AI844038Mm.7919 0.0322 0.0323 0.0399 0.0339 0.0511 0.0232 HGF-regulated tyrosinekinase substrate AI838614 Mm.14802 0.0412 0.0399 0.0281 0.0331 0.04070.0464 H19 fetal liver mRNA AI849859 Mm.3126 0.0204 0.0173 0.0375 0.03230.0641 0.0296 four and a half LIM domains 1 AI837752 Mm.43278 0.03460.0221 0.0848 0.0314 0.0460 0.0454 olfactomedin related ER localizedprotein AI841798 Mm.4871 0.0533 0.0983 0.1831 0.0273 0.0217 0.0219tissue inhibitor of metalloproteinase 3 AI838607 Mm.4159 0.0277 0.03010.0276 0.0268 0.0559 0.0602 thrombospondin 1 AI842703 Mm.147387 0.02840.0297 0.0391 0.0200 0.0205 0.0253 procollagen, type III, alpha 1

[0237] Differentially Expressed Genes (Using Progressively LessAmine-Labeled RNA)

[0238] Since 95 genes (Table 3) were 3-fold over- or under-expressedwhen C2 and 3T3 cell profiles are compared using an optimal amount oftotal RNA (see above), it was of interest to determine how many of thesegenes remained 3-fold changed when progressively smaller amounts of RNAwere labeled with the amine-modified primer method. C2 and NIH 3T3 RNAsamples were diluted in parallel, labeled with Cy3 and Cy5,respectively, the products mixed, and one 9568-element array probed perdilution. Most of the original 95 differentially expressed genes (Table3) were identified (i.e., ratios≧3 or ≦⅓ between signals from the twocell lines) when 5 μg (95 genes), 2.5 μg (90 genes), and 1 μg (87 genes)of total RNA was labeled. The number of other genes not among theoriginal 95 genes identified, but which were 3-fold changed, was fairlysmall (an average of 12).

[0239] With 0.5 μg of total RNA, only 72 of the differentially expressedgenes were found, but the number, 11, of extraneous genes remained low.When probe was made from 0.25 μg or 0.1 μg of total RNA, there was afurther decrease in differentially expressed genes detected (53 and 58,respectively), and a marked increase in false positives (71 and 97,respectively).

[0240] Analysis of Consistency of Over- or Under-expressed Genes

[0241] To determine how many genes will survive the above comparisonwhen a fourth microarray is examined using the same experimentalconditions, a model was studied. In the model, a log-transformed geneexpression ratio, w=logt, was assumed to be normally distributed with astandard deviation of σ. For this model, the probability of observing aratio measurement greater than 3.0 in one experiments is,$\begin{matrix}{p = {{P_{\mu = w}\left( {x > {\ln \quad 3}} \right)} = {\int_{\ln \quad 3}^{\infty}{\frac{1}{\sqrt{2{\pi\sigma}}}e^{- \frac{{({x - w})}^{2}}{2\sigma^{2}}}{x}}}}} & (1)\end{matrix}$

[0242] where 1n(•) denotes the natural logarithm. For a ratiomeasurement to be greater than 3 in all of two, three or fourexperiments, the probabilities are simply p², p³, and p⁴, respectively.It is further assumed that within a confined ratio region [l₁, l₂],where l₁≦3≦l₂, there is equal probability for all ratio values, orp_(r). Thus, the probability that any gene ratio within the region l₁ tol₂ is greater than 3 is given by, $\begin{matrix}{p = {{\int_{l_{1}}^{l_{2}}{p_{r}{P_{\mu = w}\left( {x > {\ln \quad 3}} \right)}\quad {w}}} = {P_{r}{\int_{w = l_{1}}^{l_{2}}{\int_{x = {\ln \quad 3}}^{\infty}{\frac{1}{\sqrt{2{\pi\sigma}}}e^{- \frac{{({x - w})}^{2}}{2\sigma^{2}}}{x}{w}}}}}}} & (2)\end{matrix}$

[0243] The difference in the expected number of genes in 3 consistentexperiments and 4 consistent experiments is,

[0244] $\begin{matrix}{n = {N\left\lfloor {{\int_{1}^{l_{2}}{{P_{r}\left\lbrack {P_{\mu = w}\left( {x > {\ln \quad 3}} \right)} \right\rbrack}^{3}{w}}} - {\int_{1}^{l_{2}}{{p_{r}\quad\left\lbrack {P_{\mu = w}\left( {x > {\ln \quad 3}} \right)} \right\rbrack}^{4}{w}}}} \right\rfloor}} & (3)\end{matrix}$

[0245] where N is the total number of genes within the region of [l₁,l₂]. The result for expression ratio less than ⅓ can be similarlyderived. Given that the number of consistent genes were known (m=95 inthis study), $\begin{matrix}{n = {m\left\lfloor {1 - \frac{\int_{l_{1}}^{l_{2}}{\left( \left\lbrack {P_{\mu = w}\left( {x > {\ln \quad 3}} \right)} \right\rbrack \right)^{4}{w}}}{\int_{l_{1}}^{l_{2}}{\left( \left\lbrack {P_{\mu = w}\left( {x > {\ln \quad 3}} \right)} \right\rbrack \right)^{3}{w}}}} \right\rfloor}} & (4)\end{matrix}$

[0246] To numerically evaluate the above equation, a typical σ=0.07 waschosen, which can be estimated from the duplicated elements printed onthe array. A typical region [l₁, l₂] was also selected, for thethreshold under consideration, to be [1n(2.0), 1n(4.5)]. For m=95 (3fold changes were lumped together since Eq. 4 for over-expression andunder-expression are identical). On this basis n=3.6. If σ=0.14, whichis the typical variation derived from the self-on-self experiment,n=8.6. Therefore, when a 4^(th) microarray in the same experimentcondition is introduced, among 95 consistently 3-fold differentiallyexpressed genes, 4 to 9 genes are expected to be dropped due to randomvariation of the microarray assay. In other words, 90 and 87 genesobtained from 2.5 μg and 1 μg were within the expectation, thus theirexperiment conditions should be comparable, although the amount of RNAused to make probe was different. For less input RNA (from which 72 orless genes in the differentially expressed class were detected), thenumber is far below that expected, and it is concluded that insufficientRNA was employed.

[0247] Modified Random Primer Labeling Shows No Cyanine Label Bias

[0248] In all of the reported studies with the new labeling techniques,only 1-5 μg or less total RNA was used as template. In spite of the lowamount of total RNA used, this system produces highly reliable andconsistent data.

[0249] To test the labeling and hybridization signal reliability of thenew labeling method, the same amount RNA was labeled (5 μg mouse C2 cellline total RNA) with two different dyes (Cy3 and Cy5) to generate Cy5and Cy3-labeled probes. The two probes were hybridized to the arrays andscanned (photomultiplier tube (PMT) voltages of 600 and 550 for Cy5 andCy3, respectively. Scatter plots of log intensity Cy5 signal versus logintensity Cy3 signal and log (Cy5/Cy3) versus Average log intensity areshown. Data shows (FIG. 3A) that the two probes generated similar signalintensities though they were labeled by two different dyes.

[0250] Cy5 and Cy3-labelled probes were also prepared from 5 μg and 1 μgof total C2 RNA, respectively. PMT voltages of 600 and 580 were used toscan the Cy5 and Cy3 channels. These signals were strongly correlated(FIG. 3B).

[0251] A recent study using traditional labeling techniques (Taniguchiet al., Genomics 71, 34-39, 2001) clearly showed the inconsistency oflabeling and hybridization results from the reverse combination of dyes,due to the bias of dye labeling. In that study, a notably largerquantity of template RNA was required for Cy5 labeling when thetraditional direct labeling method was used. The modified random primerlabeling system reported herein overcomes this labeling bias.

[0252] P1 Versus P2, Same Amount of Template RNA

[0253] Another experiment was carried out to compare hybridizationsignals from probe labeled with amine modified random primer P2 andregular random primer P1, using 5 μg total RNA from mouse C2 cell linefor both labeling methods and both Cy3 and Cy5 labeling.

[0254] The two probes labeled using two different primers werehybridized to each of two identical arrays on the same slide, asdescribed above. The slide was then scanned at same laser power and PMTlevel (620 volts and 600 volts for the Cy5 and Cy3 channels,respectively). The images were processed and analyzed withIPLab/ArraySuite. The hybridization intensity from the array hybridizedwith probe labeled with primer P2 were substantially stronger than theintensity achieved from probe labeled with primer P1.

[0255] These comparison data were quantified and indicate thathybridization intensities using P2 labeling were at least 2.5 foldhigher than P1. Amine modified random primer P4 showed similar results.Thus, with more amine groups being incorporated into the probes usingthe modified random primers, the fluorescent signals are demonstrated tobe much higher when using an equivalent amount of starting template.

[0256] This reveals that, when comparing the traditional random primer(P1) with an amine modified random primer (P2), the signal fromincorporating a primer with a single amine (—NH₂) group into each cDNA(using P2) is roughly equivalent to that achieved when amine modifiedbase is included only in the RT reaction (using P1). Thus, roughly only1-2 amine labeled nucleotides are incorporated by RT per strandsynthesized; this suggests that synthesis may cease once a singlemodified nucleotide is incorporated. Therefore, one strategy forincreasing incorporation is not by adding amine- or dye-modifiednucleotides in the RT reaction, but by adding additional modificationsto the primer. For this reason, also provided are additional modifiedprimers (SEQ ID NOs: 4-9, for instance) comprising two (or more)modified bases (e.g., amine-modified bases), which optionally may beseparated by 0-5 inosine residues. Signal intensity from the labelmolecule may vary depending on the spacing between multiple modifiedbases within a single primer.

[0257] Sensitivity and Clone Detection

[0258] The modified primer labeling method increased hybridizationsensitivity as well. Starting with the same amount of template RNA,probes labeled using amine modified random primer P2 could detect about60 genes that were not detectable with probes labeled using regularrandom primer P1.

[0259] A recent study (Taniguchi et al., Genomics 71, 34-39, 2001)demonstrated that some genes were not detectable using standard DNAmicroarrays when compared with conventional Northern blot analysis. Thisdefect in the traditional labeling method may be overcome using themodified random primer labeling methods disclosed herein.

[0260] In distinct contrast to prior labeling methods, the modifiedprimer labeling methods, as demonstrated here with amine modified randomprimer, produce significant signals and increases sensitivities, whetherthe probe is labeled with Cy3 or Cy5. Much less RNA is required formaking high quality probes and there is no bias of dye incorporationusing same amount of RNAs for both Cy3 and Cy5 labeling.

Example 4 Amplification Coupled With Amine Modified Random PrimerLabeling (Method 1)

[0261] The disclosed amine modified random primers can also be used withT7-mediated amplification of transcript, to further reduce the amount ofstarting material necessary to produce a hybridization probe. This canbe carried out using the following protocol:

[0262] I. cDNA Synthesis

[0263] First strand synthesis is carried out using the followingPrimer-RNA mixture: Primer-RNA mix Total RNA (less than 1 μg) 1-5 μlDEPC water variable T7 - Oligo dT primer (100 pm/μl)   1 μl Total  10 μl

[0264] This mixture is incubated at 70° C. for 10 minutes, then chilledon ice 10 minutes to facilitate annealing of the primer to the template.

[0265] To each reaction is then added 10 μl of the following reversetranscription mixture: RT mix Component μl For 10 reactions (10.2 fold)5X first strand buffer 4 40.8 10 mM dNTPs 1 10.2 DTT (0.1 M) 2 20.4 DEPCwater 1 10.2 SSII RT 2 30.6 Total 10

[0266] The first strand of cDNA is synthesized by incubating the tubesat 42° C. for 2 hours.

[0267] To initiate second strand synthesis, the following reagents aremixed with a first strand synthesis reaction: RNase-free water  91 μl 5Xsecond strand buffer  30 μl 10 mM dNTPs  3 μl E. coli DNA ligase  1 μlE. coli DNA polymerase I  4 μl RNase H  1 μl Total (including firststrand reaction) 150 μl

[0268] The reaction mixture is then incubated at 16° C. for 2 hours. A 2μl aliquot of T4 DNA polymerase is added, and the mixture incubated at16° C. for 5 minutes. The reaction is stopped by adding 10 μl of 0.5 MEDTA (pH 8.0)

[0269] The double stranded cDNA (ds cDNA) is then extracted, forinstance using Phase Lock Gel (PLG) extraction, using the manufacturer'sinstructions. To make it ready for use, the PLG tube is pelleted bycentrifuging for 30 seconds at maximum speed in a microfuge. The ds cDNA(approximately 162 μl) is mixed with an equal volume ofPhenol-Chloroform-IAA (162 μl) and vortexed. All of this mixture isadded to the PLG tube, and the tube centrifuged for two minutes atmaximum speed.

[0270] The resulting ds cDNA preparation can be further cleaned up usingfor instance, a Microcon 100 concentrator from Amicon. The Microcon 100is filled with 400 μl dd-H₂O, and the top aqueous layer from above PLGextraction transferred into it. The column is then spun at maximum speedfor about 2 minutes (or until about 20 μl left), and the flow-throughdiscarded. This washing process is repeated twice more with 500 μldd-H₂O. The concentrated and cleaned ds DNA sample is collected byinverting the tube and spinning at 5000 rpm for 5 minutes. The resultantsample is dried in a vacuum centrifuge, and re-suspended in 4.5 μl ofRNase-free water.

[0271] II. In Vitro Transcription

[0272] Double-stranded cDNA produced as above is then used in an invitro transcription reaction, using for instance an Ambion in vitrotranscription (IVT) kit, as follows:

[0273] The IVT reaction comprises the following: Ambion T7 10X ATP   2μl Ambion T7 10X GTP   2 μl Ambion T7 10X UTP   2 μl Ambion T7 10X CTP  2 μl RNase-free water 3.5 μl ds DNA synthesized above 4.5 μl 10X T7transcription buffer   2 μl 10X T7 enzyme mix   2 μl Total  20 μl

[0274] The transcription reaction is incubated at 37° C. for six hours

[0275] In vitro transcribed RNA made in this manner can be cleaned up,for instance, using Qiagen RNeasy mini columns and protocols as suppliedby the manufacturer, essentially as follows:

[0276] In 1.5 ml tube, the following reagents are mixed: RNase freewater  80 μl IVT reaction  20 μl Buffer RLT (supplied with kit) 350 μl100% EtOH 250 μl

[0277] The mixture (700 μl) is vortexed gently, placed in an RNeasycolumn, and incubated for two minutes to provide time for the RNA tobind to the column. The column is then centrifuged at 2000 rpm for 5minutes, and the flow-through reserved. The column is washed (twice)with 500 μl of RPE (with EtOH added), and centrifuged at 10,000 rpm for1 minute. The column is then centrifuged at maximum speed for 1 minuteto remove any remaining fluid, and placed in a new 1.5 ml collectiontube. RNase-free water (30 μl) is added, and the tube incubated for 1-2minutes. The column is centrifuged at 5000 rpm for 5 minutes, then10,000 rpm for 30 seconds, and the eluate is collected. The elutionprocess is repeated with an additional 30 μl of RNase-free water, togive a final elution volume of approximately 60 μl. The copy RNA (cRNA)yield can be quantitated by measuring its optical density (OD) usingstandard techniques.

[0278] III. Labeling With Modified Random Primer Using cRNA as Template

[0279] cRNA produced as above can be used as the template for productionof labeled probe molecules using the modified (e.g., amine modified)random primers provided herein.

[0280] A 17 μl aliquot of the RT mix (6 μl 5×first strand buffer(provided with SSII RT), 6 μl 5×aa-dUTP/dNTPs, 3 μl 0.1M DTT, and 2 μlSuperScript II Reverse transcriptase (SSII RT; GIBCO BRL LifeTechnologies, Rockville, Md.)) is added to each primer-RNA mix, for atotal volume of 30 μl, and the sample incubated at 42° C. for two hoursto permit reverse transcription of the cDNA.

[0281] The reverse transcription reaction was stopped by the addition of10 μl of 0.5M EDTA. RNA was degraded by adding 10 μl of 1N NaOH, andincubating the sample at 65° C. for 30 minutes. The reaction was thenneutralized by adding 10 μl of 1M HCl.

[0282] The neutralized cDNA sample was cleaned up using a MicroCon 100concentrator cartridge (Millipore, Bedford, Mass.). The cartridge wasprimed with 450 μl of ddH₂O, then the neutralized reaction solution wasadded and the cartridge spun at 13,000 rpm for about 3 minutes. Theflow-through was discarded, and the cDNA on the cartridge was washedtwice with 500 μl of ddH₂O. The cDNA sample was eluted from thecartridge, and dried in a vacuum centrifuge.

[0283] IV. Coupling, Quenching and Cleanup

[0284] The cDNA sample is resuspended in 4.5 μl water. Pre-driedaliquots of monofunctional NHS-ester Cy3 or Cy5 dye (prepared as inExample 2) resuspended in 4.5 μl of 100 mM NaBicarbonate Buffer (pH9.0). One aliquot of cDNA is mixed with one aliquot of Cy3 or Cy5, andthe samples are incubated at RT for 1 hour in the dark to couple thefluorescent dye to the modified cDNA.

[0285] The fluorescence is quenched by adding 4.5 μl of 4M hydroxylamineto the dye-coupled cDNA, and incubating the mixture at RT for 30 minutesin dark. The labeled sample is cleaned up using a Qiagen Qia-quick PCRpurification kit (Qiagen, Valencia, Calif.) as described in Example 2.

[0286] Hybridization to microarrays and analysis of the resultant dataare carried essentially as described above in Example 3.

Example 5 Amplification Coupled With Amine Modified Random PrimerLabeling (Method 2)

[0287] In another embodiment, asRNA is amplified using the followingprotocol. Total RNA is isolated from a biological sample, such as afresh or preserved cell or tissue sample or an aliquot of cells grown inculture. By way of example, total RNA is isolated using a Qiagen midikit (Cat. #75142) following the instructions provided by themanufacturer. Alternatively, Trizol extraction (Gibco BRL Cat.#15596-026) could also be used (following the procedures provided by themanufacturer). The total RNA is then resuspended or eluted in DEPCwater.

[0288] First strand cDNA synthesis is carried out as follows: In a PCRreaction tube, 0.001-3 μg total RNA is mixed in 9 μl DEPC H₂O with 1 μl(0.01-0.5 μg/μl) oligo dT₍₁₅₎-T7 primer (SEQ ID NO: 11) and heated to70° C. for three minutes, then cooled to room temperature. To this isthen added the following reagents (which can be made into a “mastermix”for multiple samples):

[0289] 4 μl 5×First strand buffer (provided with Superscript II kit)

[0290] 1 μl (0.1-0.5 μg/μl) TS (template switch) oligo primer (SEQ IDNO: 3)

[0291] 2 μl 0.1M DTT

[0292] 1 μl RNasin (Promega Cat. #N2111)

[0293] 2 μl 10 mM dNTP (Pharmacia Cat. #27-2035-02)

[0294] 2 μl Superscript II polymerase (Gibco BRL Cat. #18064-071)

[0295] The reaction is then incubated 42° C. for 90 minutes, forinstance in a thermal cycler.

[0296] Second strand synthesis is carried out by adding the followingreagents to each cDNA reaction tube:

[0297] 106 μl of DEPC H₂O

[0298] 15 μl Advantage PCR buffer

[0299] 3 μl 10 mM dNTP

[0300] 1 μl of RNase H (2U/μl, Gibco BRL Cat#18021-071)

[0301] 3 μl Advantage Polymerase (CLONTECH Cat#8417-1)

[0302] The samples are then incubated at 37° C. for five minutes todigest mRNA, 94° C. for two minutes to denature, 65° C. for one minutefor specific priming, and 75° C. for 30 minutes for extension of thesecond strand. The reaction is stopped by adding 7.5 μl 1M NaOH solutioncontaining 2 mM EDTA and incubating at 65° C. for 10 minutes toinactivate enzyme.

[0303] The double stranded (ds) cDNA can be cleaned up as follows: A 1μl aliquot of Linear Acrylamide (0.1 μg/μl, Ambion Cat. #9520) is addedto each sample. The sample is then extracted by adding 150 μl Phenol:Chloroform: Isoamyl alcohol (25:24:1) (Boehringer Mannheim Cat. #101001)to each ds cDNA tube and mixing well by pipetting. It is important to becareful not to spill or contaminate the sample. The slurry solution isthen transferred to Phase lock gel tube (5′-3′ Inc. Cat. #p1-257178) andspun at 14,000 rpm for five minutes at room temperature. The aqueousphase is transferred to RNase/DNase-free tube and 70 μl of 7.5M ammoniumacetate (Sigma Cat#A2706) added, followed by 1 ml 100% ethanol. Thistube is centrifuged at 14,000 rpm for 20 minutes at room temperature topellet the nucleic acid. The resultant pellet is washed twice with 500μl 100% ethanol and spun down at maximum speed for eight minutes.Finally, the ds cDNA pellet is air dried and resuspended in 70 μl DEPCH₂O.

[0304] Bio-6 Chromatograph columns (Bio-Rad Cat. #732-6222) are preparedby washing the columns with 700 μl DEPC H₂O three times and spinning at700×g for two minutes at room temperature. (It may be important to shakethe washed column well before draining to get rid of airbubbles—otherwise it drains very slowly.) When opening the column, anygel in the underside of the cap is aspirated off to preventcontamination. Also, the collection tubes provided with Bio-6 columnsare not RNase-free; the samples should be collected in RNase-free tubes.

[0305] For each sample, 70 μl is loaded onto the center of the columnand the column spun at 700×g for four minutes. The sample is then driedin a vacuum centrifuge and resuspended in 8 μl DEPC water.

[0306] Using this double-stranded cDNA, in vitro transcription (IVT) isperformed using an Ambion T7 Megascript Kit (Cat. #1334). For eachsample, the following reaction mixture is made:

[0307] 2 μl of each 75 mM NTP (A, G, C and UTP)

[0308] 2 μl reaction buffer

[0309] 2 μl enzyme mix (RNase inhibitor and T7 phage polymerase)

[0310] 8 μl ds cDNA (produced as described herein)

[0311] The reactions are then incubated at 37° C. for six hours topermit transcription.

[0312] The asRNA produced is then purified using TRIzol reagent(GibcoBRL, Cat. #15596). To each IVT reaction is added 1 ml of TRIzolsolution, and the tubes are mixed well. 200 μl of chloroform is thenadded per 1 ml TRIzol solution, and the samples mixed by inverting for15 seconds. They are then incubated at room temperature for 2-3 minutes,and centrifuged at 12,000g for 15 minutes at 4° C. The aqueous phase isthen transferred to a new RNase free tube and 500 μl of isopropylalcohol added per 1 ml TRIzol reagent to precipitate the nucleic acids.The samples are incubated at room temperature for 10 minutes and thencentrifuged at 14,000 rpm for 15 minutes. The resultant pellet is washedtwo times with 1 ml 70% ethanol in DEPC-treated water, the pellet airdried and quickly resuspended in 20 μl DEPC-treated water. (Over-driedRNA is difficult to dissolve into water). RNA concentration can bechecked and quality estimated by measuring OD₂₆₀ and OD₂₆₀/₂₈₀ usingstandard techniques.

[0313] An RNAeasy mini kit also could be used to recover the asRNA (butthe recovery of asRNA may be lower that that achieved with the TRIzolmethod.)

[0314] The asRNA may be subjected to a second round of amplification,though this is not necessary in all embodiments. By way of example,asRNA (0.5-1 μg) produced as above is mixed in 9 μl DEPC H₂O with 1 μl(2 μg/μl) random hexamer (i.e., dN₆) and heated to 70° C. for threeminutes, then cooled to room temperature. The following reagents arethen added:

[0315] 4 μl 5×First strand buffer

[0316] 1 μl (0.5 μg/μl) oligo dT-T7 primer

[0317] 2 μl 0.1M DTT

[0318] 1 μl RNasin (Promega Cat. #N2111)

[0319] 2 μl 10 mM dNTP (Pharmacia Cat. #27-2035-02)

[0320] 2 μl Superscript II (SS II) (Gibco BRL Cat. #18064-071)

[0321] The samples are then incubated at 42° C. for 90 minutes. Theresultant single-stranded cDNA then can be subjected to second strandsynthesis and cleanup similarly to that described above. By way ofexample, the ds cDNA is then resuspended in 16 μl of DEPC treated water.

[0322] Second round in vitro transcription (IVT) proceeds using thefollowing reaction mixture:

[0323] 4 μl of each 75 mM NTP (A, G, C and UTP)

[0324] 4 μl reaction buffer

[0325] 4 μl enzyme mix (RNase inhibitor and T7 phage polymerase)

[0326] 16 μl ds cDNA

[0327] Each reaction is incubated at 37° C. for six hours, and the asRNApurified using TRIzol reagent, as described above.

[0328] asRNA amplified from the second IVT then can be converted intocDNA using modified random primers as provided herein and reversetranscription, for instance using the following reaction:

[0329] 6 μg of asRNA (1 μg/μl)

[0330] 2 μl of modified random primer (8 μg/μl)

[0331] 14 μl of DEPC treated water

[0332] Samples are heated to 70° C. for three minutes and then put onice. Then, the following reagents are added:

[0333] 8 μl of 5×first strand buffer

[0334] 4 μl of 10 mM dNTP (with or without the addition of aa-dNTP asdescribed herein)

[0335] 4 μl of 0.1M DTT

[0336] 2 μl of RNasin

[0337] 3 μl of Superscript II

[0338] The samples are then incubated at 42° C. for 90 minutes. Thereactions are stopped by adding 5 μl of 0.5M EDTA with 10 μl of 1M NaOHand heating to 65° C. for 10 minutes, which hydrolyzed the asRNA andinactivated the enzymes. The pH of the samples is neutralized by adding25 μl of 1M Tris pH 7.5.

[0339] Target nucleic acids may be purified (precipitated) as follows:To each sample is added 30 μl of ammonium acetate and 500 μl 100%ethanol, and the samples are mixed and incubated at −20° C. for 15minutes. Samples are centrifuged at 13,000 rpm at 4° C. for 20 minutes,and the resultant pellet washed twice with 500 μl of 70% ethanol. Thepellet is then completely dried using a Speedvac, and the purified cDNAresuspended in 12.5 μl of 3×SSC; in some embodiments, to get a strongersignal the cDNA is resuspended in a smaller volume. Resuspended cDNA canbe stored at −20° C. prior to labeling with a detectable molecule, suchas a Cy3 or Cy5.

Example 6 RNA Amplification With T3N9 Primers Coupled With AmineModified Random Primer Labeling (Method 3)

[0340] The disclosed primer modification (such as amine modification)can be used with T3N9 primer-mediated amplification of transcript toproduce a collection of RNA species. An advantage of using the T3N9primer is that, unlike transcripts generated with random hexamers andT7-oligo dT primers, transcripts generated with T3N9 primers aresubstantially less 3′ biased. As a result, the length of T3N9primer-mediated transcripts tends not to decrease with each round ofamplification. By way of example, amplification using T3N9 primers canbe carried out using the following protocols:

[0341] I. RNA Production

[0342] Amplified RNAs were prepared either from total RNA sources ordirectly from cells.

[0343] If starting with cells, BCBL1 and 293 cells were collected andwashed in cold 1×PBS (Invitrogen, Carlsbad, Calif.). The cells werecounted and diluted to a density of 5000 cells/ml. Two μl of cells (˜10cells) were added to a 0.5 ml tube containing a mixture of 6 μl of5×first strand buffer (Invitrogen, Carlsbad, Calif.), 31 μl ofRNase-free water (Invitrogen, Carlsbad, Calif.), and 1 μl of RNaseinhibitor (Promega, Madison, Wis.). The cells were broken apart bysonication. After spinning at 13,000 rpm at 4° C. for 15 minutes, thesupernatant was transferred to a 0.2 ml PCR tube and concentrated to 23μl with a SpeedVac (Thermo Savant, Holbrook, N.Y.). In order to digestthe genomic DNA, 0.5 μl of DNase I (Ambion, Austin, Tex.) was added tothe sample, then incubated for 30 minutes at 37° C. The DNase I wasinactivated by incubating the sample at 75° C. for 5 minutes.

[0344] If starting with total RNA, human BCBL1 and 293 cells werecollected and total RNA was extracted using TRIzol reagent fromInvitrogen Life Technologies (Carlsbad, Calif.) following themanufacturer's instructions. Two μl of total RNA (0.5 μg) was added in a0.2 ml PCR tube containing 6 μl of 5×first strand buffer, 31 μl ofRNase-free water, and 1 μl of RNase inhibitor. The sample wasconcentrated to 23 μl before initiating the first strand cDNA synthesis.

[0345] II. cDNA Synthesis

[0346] T7-oligo dT primer (SEQ ID NO: 13) from Operon (Alameda, Calif.)(1 μl, at a concentration of 100 pmol/μl) was added to 23 μl of totalRNA or the RNA derived from the 10 cells, as described above. The RNAwas denatured at 70° C. for 10 minutes and chilled, on ice, for 10minutes. 1 μl of 10 mM dNTPs (Amersham Pharmacia, Piscataway, N.J.), 3μl of 0.1 mM DTT (Invitrogen, Carlsbad, Calif.) and 2 μl of SuperScriptII reverse transcriptase (Invitrogen, Carlsbad, Calif.) were added tothe tube, and the reaction mixture was incubated at 42° C. for 2 hoursto carry out first strand cDNA synthesis.

[0347] For second strand cDNA synthesis, 81 μl of RNase-free water, 30μl of 5×second strand buffer (100 mM Tris-HCl, pH 6.9; 450 mM KCI; 23 mMMgCI₂; 0.75 mM beta-NAD⁺; and 50 mM (NH₄)₂SO₄), 3 μl of 10 mM dNTPs, 1μl of E. coli DNA ligase (Invitrogen, Carlsbad, Calif.), 4 μl of E. coliDNA polymerase I (Invitrogen, Carlsbad, Calif.), and 1 μl of RNase H(Invitrogen, Carlsbad, Calif.) were added to bring the total volume ofthe sample to 150 μl. The reaction was then incubated for 2 hours at 16°C. Following the incubation, 2 μl of T4 DNA polymerase (Invitrogen,Carlsbad, Calif.) was added to the sample, followed by a 5 minuteincubation at 16° C.

[0348] Phase Lock Gel (Eppendorf, Westbury, N.Y.) andphenol-chloroform-IAA (Invitrogen, Carlsbad, Calif.) were used toextract the cDNA using the manufacturer's protocol. The sample was thenapplied to a MicroCon-30 column (Millipore, Bedford, Mass.) to furtherclean and concentrate the cDNA. The cDNA was dried in a SpeedVac andresuspend in 4.5 μl of RNase-free water.

[0349] III. RNA Amplification

[0350] First round amplified RNA was then transcribed from thedouble-stranded cDNA with MEGAscript T7 kit (FIG. 4) (Ambion, Austin,Tex.), according the manufacturer's instructions, followed by clean-upwith RNeasy Mini kit (Qiagen, Valencia, Calif.).

[0351] The second and subsequent rounds of amplification were carriedout using two different methods (FIG. 4). One method was essentially asdescribed by Wang et al., Nat. Biotechnol. 18, 457-459 (2000).Specifically, 0.5-1 μg first round amplified RNA was mixed with 1 μl ofrandom hexamer (dN6) (2 μg/μl) in 9 μl DEPC water. The mixture washeated to 70° C. for 3 minutes, then cooled to room temperature. Thefollowing reagents were added to the mixture: 4 μl 5×first strand buffer(provided with Superscript II), 1 μl (0.5 μg/μ) oligo dT-T7 primer, 2 μl0.1M DTT, 1 μl RNAsin (Promega Cat#N2111), 2 μl 10 mM dNTP (PharmaciaCat#27-2035-02), and 2 μl Superscript II (SS II) (Gibco BRLCat#18064-071). The mixture was incubated at 42° C. for 90 minutes.Second strand cDNA synthesis and double stranded cDNA cleanup wereperformed as described above. In the second round of in vitrotranscription, 40 μl of the in vitro transcription reaction mixture wasused instead of 20 μl. RNA isolation followed, as described above.

[0352] The second method of second and subsequent rounds ofamplification used a custom designed T3N9 primer (SEQ ID NO: 12)(Invitrogen, Carlsbad, Calif.) for priming both the first strand cDNA.Specifically, 17 μl of first round amplified RNA was mixed with 1 μl ofT3N9 (100 pm/μl) and the mixture was incubated at 70° C. for 10 minutesthen chilled, on ice, for 10 minutes. The following reagents were thenadded to the mixture: 6 μl 5×first strand buffer, 1 μl of 10 mM dNTPs(Amersham Pharmacia, Piscataway, N.J.), 3 μl of 0.1 mM DTT (Invitrogen,Carlsbad, Calif.) and 2 μl of SuperScript II reverse transcriptase(Invitrogen, Carlsbad, Calif.) were added to the tube, and the reactionmixture was incubated at 42° C. for 2 hours to carry out first strandcDNA synthesis. Second strand cDNA synthesis, double stranded cDNAclean-up, and subsequent in vitro transcription were performed asdescribed above.

[0353] IV. Probe Labeling Using Amine Modified Random Primers

[0354] The amplified RNA can be used as a template for production oflabeled probe molecules using the modified (e.g., amine modified)primers, as described in Examples 4 and 5 above. Five μg of total RNA or2 μg of amplified RNA (5 μg for the amplified RNA obtained directly fromcells) were used for labeling the cDNA probes.

[0355] V. cDNA Microarrays

[0356] Amplified RNA generated from 1-4 rounds of amplification with theT3N9 primer, as described above, and total RNA, were obtained from humanBCBL1 and 293 cell lines. Using the total RNA or the amplified RNA astemplates, cDNA probes were generated with the amine modified randomprimers, as described in Examples 4 and 5, above. The probes were thenhybridized to the microarrays as follows: the cDNA probes were partiallydried in a vacuum centrifuge to a volume of 17 μl and to the DNA wasadded 1 μl of poly A (8 mg/ml), 1 μl of Cot-1 DNA (10 mg/ml) and 1 μl ofyeast tRNA (4 mg/ml). The probe mixture was denatured at 98° C. for 2minutes, chilled on ice and 20 μl of the probe mixture was mixed with 20μl of 2×F-Hybridization buffer (250 μl of 100% formamide, 250 μl of20×SSC, 10 μl of 10% sodium dodecyl sulfate). An aliquot of the mixture(35 μl) was applied to arrays. The arrays were covered with 22×60 mmcoverslips and then incubated overnight, in a water bath, at 42° C.Following the incubation, the cover-slips were removed from the arrayswhile they were soaking in pre-wash buffer (2×SSC, 0.1% sodium dodecylsulfate) and the arrays were washed for 5 minutes at room temperature infirst wash buffer (0.5×SSC, 0.01% sodium dodecyl sulfate) followed by awash with second wash buffer (0.06×SSC) for 5 minutes at roomtemperature. The arrays were dried by spinning them in a centrifuge at800 rpm for 2 minutes.

[0357] All experiments used 6500 element human cDNA arrays. The ratioswere determined by comparing the intensities, captured with a laserscanner, of the BCBL1 and 293 cell lines (FIG. 5). A strong correlationwas observed between (FIG. 5A) total RNA/first round amplification,(FIG. 5B) first/second round amplification, (FIG. 5C) second/third roundamplification, and (FIG. 5D) third/fourth round amplification, withR²=0.8256, 0.9001, 0.8561, and 0.8539, respectively. A good correlationwas also demonstrated in FIG. 5E after three rounds of amplificationusing the T3N9 primers (R²=0.8018) compared to the standard method, asshown in FIG. 5F, that uses random hexamers and T7-oligo dT primers(R²=0.4818).

[0358] VI. Differentially Expressed Genes

[0359] In one specific experiment, cultured mouse C2 and NIH 3T3 cellswere diluted to a density of 10 or 100 cells per sample. First strandcDNA synthesis directly from cells, second strand cDNA synthesis and RNAamplification were performed as described above. After three rounds ofamplification, approximately 20 μg of amplified RNA was obtained. Halfof the amplified RNA was used in the labeling reaction. The microarrayexpression patterns were similar between the total RNA and aRNA and theRNA amplified from 10 and 100 single cells. Genes with a 3-fold orgreater difference in expression were identified (73 genes) which wascomparable to the number of genes identified (90 genes) with total RNA.The most differentially expressed genes are listed in Table 4. TABLE 4Total RNA 10cellAmp3rd Clone ID Description 18.3218 9.6548 AI849214 wheyacidic protein 6.6261 4.1602 AI836864 forkhead box G1 6.2954 3.375AI838361 Mus musculus 10 days embryo cDNA, RIKEN full-length enrichedlibrary, clone: 2610305D13, full insert 5.528 6.2676 AI843085 RIKEN cDNA5730403B10 gene 5.5015 4.6931 AI842716 cytochrome P450, 51 5.2438 4.4078AI847098 ERO1-like (S. cerevisiae) 5.225 7.4932 AI846827 Mus musculus,Similar to oxidation resistance 1, clone MGC: 7295, mRNA, complete cds4.0612 12.1508 AI840688 transketolase 3.7839 6.6078 AI852317 N-mycdownstream regulated 1 3.7376 3.2635 AI843677 Erbb2 interacting protein3.715 3.6818 AI844828 glycine transporter 1 3.2641 3.0637 AI847571matrin 3 3.0989 8.5314 AI841304 EST 3.0926 11.1423 AI838612 glutathioneS-transferase, mu 2 3.0799 3.7138 AI847962 transmembrane 4 superfamilymember 2 3.0628 3.2787 AI839363 mammary tumor integration site 6 0.29260.0848 AI846190 ATPase, H+ transporting, lysosomal (vacuolar protonpump), alpha 70 kDa, isoform 2 0.2816 0.3131 AI838302 Cd63 antigen0.2799 0.1641 AI845774 transmembrane 4 superfamily member 1 0.27930.2697 AI835620 No Data 0.2668 0.1074 AI851985 RIKEN cDNA 2610024P12gene 0.264 0.1284 AI840752 cAMP responsive element binding protein 30.2615 0.2032 AI838653 RIKEN cDNA 2610001E17 gene 0.2547 0.1201 AI844356esterase 10 0.2365 0.1955 AI851647 ESTs, Weakly similar to SH3B_MOUSESH3 DOMAIN-BINDING GLUTAMIC ACID-RICH PROTEIN (SH3BGR PROTEIN) 0.23360.3206 AI842654 lymphocyte antigen 6 complex 0.2151 0.1776 AI836265 ESTs0.2107 0.1573 AI839057 No Data 0.2013 0.185 AI842847 tissue inhibitor ofmetalloproteinase 0.1946 0.1997 AI853210 procollagen, type IV, alpha 10.1791 0.3161 AI834944 RIKEN cDNA 5530400B01 gene 0.1639 0.1518 AI849378MARCKS-like protein 0.1605 0.1248 AI838551 prostaglandin-endoperoxidesynthase 1 0.1604 0.1033 AI837494 ESTs, Weakly similar to T14318ubiquitin-protein ligase E3-alpha - mouse [M. musculus] 0.1556 0.1074AI840347 EST 0.1513 0.1874 AI842286 protein tyrosine phosphatase,receptor type, K 0.1492 0.1851 AI836264 tissue inhibitor ofmetalloproteinase 3 0.1483 0.0755 AI848096 erythrocyte protein band4.1-like 3 0.1447 0.2196 AI847958 RIKEN cDNA 2410004D18 gene 0.14220.3007 AI838351 No Data 0.1394 0.1511 AI840692 No Data 0.1356 0.1468AI839275 procollagen, type IV, alpha 1 0.1338 0.0426 AI838813 EST 0.12840.0588 AI843174 ADP-ribosylation-like factor 6 interacting protein0.1263 0.0146 AI844604 four and a half LIM domains 1 0.1258 0.2362AI842984 tenascin C 0.1166 0.0592 AI841538 Nedd4 WW-binding protein 40.1133 0.2358 AI850497 ESTs, Highly similar to LOX5 MOUSE ARACHIDONATE5-LIPOXYGENASE [M. musculus] 0.1115 0.1109 AI835201 sarcoglycan, epsilon0.1031 0.1129 AI845475 ESTs 0.0965 0.1415 AI835403 thymosin, beta 4, Xchromosome 0.0961 0.1652 AI835703 RIKEN cDNA 1810003P21 gene 0.09550.1569 AI843282 procollagen, type IV, alpha 2 0.0928 0.2414 AI840335 EST0.0926 0.1414 AI841809 SMT3 (supressor of mif two, 3) homolog 1 (S.cerevisiae) 0.0846 0.0727 AI840673 ADP-ribosylation-like factor 6interacting protein 0.0747 0.0932 AI836826 glycoprotein 38 0.0702 0.1653AI842983 EST 0.0702 0.0298 AI842681 cartilage associated protein 0.06840.2246 AI844626 RIKEN cDNA 1810003P21 gene 0.0577 0.1983 AI842554 ESTs0.0533 0.1191 AI841798 tissue inhibitor of metalloproteinase 3 0.04950.0442 AI836468 myristoylated alanine rich protein kinase C substrate0.0492 0.2086 AI839950 four and a half LIM domains 1 0.0491 0.0287AI835976 erythrocyte protein band 4.1-like 3 0.0477 0.1357 AI840633carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 0.0412 0.0659AI838614 H19 fetal liver mRNA 0.0364 0.2168 AI835609 neurofilament,light polypeptide 0.0346 0.0267 AI837752 olfactomedin related ERlocalized protein 0.0322 0.0796 AI844038 HGF-regulated tyrosine kinasesubstrate 0.0284 0.0214 AI842703 procollagen, type III, alpha 1 0.02770.0498 AI838607 thrombospondin 1 0.0204 0.0109 AI849859 four and a halfLIM domains 1

Example 7 cDNA Synthesis and RNA Amplification With T3N9 Primer CoupledWith Amine Modified Primer Labeling

[0360] The disclosed primer modification (such as amine modification)can be used with the T3N9 primer for priming cDNA synthesis from eitheran RNA or a DNA template in order to amplify RNA from small amounts ofstarting material (RNA or DNA), including single cells. The primer has aT3 polymerase recognition sequence on the 5′ end. As in Example 6, thismethod does not favor the synthesis of 3′ products. However, theadvantage of this method is that it is simpler than the one described inExample 6, as it requires the use of only a single primer throughout theprocedure.

[0361] I. RNA Production

[0362] Amplified RNAs were prepared either from total RNA sources ordirectly from cells.

[0363] If starting with cells, hypothalamic magnocellular neurons werecollected as follows: The supraoptic nucleus of the hypothalamus, one oftwo nuclei containing magnocellular neurons, were microdissected fromthe rat, following this, the cells were dissociated, and individualcells were sucked into micropipettes. Individual cells were added toseparate 0.5 ml tubes containing a mixture of 6 μl of 5×first strandbuffer (Invitrogen, Carlsbad, Calif.), 31 μl of RNase-free water(Invitrogen, Carlsbad, Calif.), and 1 μl of RNase inhibitor (Promega,Madison, Wis.). The cells were broken apart by sonication. Afterspinning at 13,000 rpm at 4° C. for 15 minutes, the supernatant wastransferred to a 0.2 ml PCR tube and concentrated to 23 μl with aSpeedVac (Thermo Savant, Holbrook, N.Y.). In order to digest the genomicDNA, 0.5 μl of DNase I (Ambion, Austin, Tex.) was added to the sample,then incubated for 30 minutes at 37° C. The DNase I was inactivated byincubating the sample at 75° C. for 5 minutes.

[0364] If starting with total RNA, mouse C2 and 3T3 cells were collectedand total RNA was extracted using TRIzol reagent from Invitrogen LifeTechnologies (Carlsbad, Calif.) following the manufacturer'sinstructions. Two μl of total RNA (0.5 μg) was added in a 0.2 ml PCRtube containing 6 μl of 5×first strand buffer, 31 μl of RNase-freewater, and 1 μl of RNase inhibitor. The sample was concentrated to 23 μlbefore initiating the first strand cDNA synthesis.

[0365] II. cDNA Synthesis

[0366] A custom designed T3N9 primer (SEQ ID NO: 12) from Invitrogen(Carlsbad, Calif.) (1 μl, at a concentration of 100 pmol/μl) was addedto 23 μl of total RNA or the RNA derived from the 1 cell, as describedabove. The RNA was denatured at 70° C. for 10 minutes and chilled, onice, for 10 minutes. 1 μl of 10 mM dNTPs (Amersham Pharmacia,Piscataway, N.J.), 3 μl of 0.1 mM DTT (Invitrogen, Carlsbad, Calif.) and2 μl of SuperScript II reverse transcriptase (Invitrogen, Carlsbad,Calif.) were added to the tube, and the reaction mixture was incubatedat 42° C. for 2 hours to carry out first strand cDNA synthesis (FIG. 6).

[0367] For second strand cDNA synthesis, 81 μl of RNase-free water, 30μl of 5×second strand buffer (100 mM Tris-HCl, pH 6.9; 450 mM KCI; 23 mMMgCI₂; 0.75 mM beta-NAD⁺; and 50 mM (NH₄)₂SO₄), 3 μl of 10 mM dNTPs, 1μl of E. coli DNA ligase (Invitrogen, Carlsbad, Calif.), 4 μl of E. coliDNA polymerase I (Invitrogen, Carlsbad, Calif.), and 1 μl of RNase H(Invitrogen, Carlsbad, Calif.) were added to bring the total volume ofthe sample to 150 μl. The reaction was then incubated for 2 hours at 16°C. Following the incubation, 2 μl of T4 DNA polymerase (Invitrogen,Carlsbad, Calif.) was added to the sample, followed by a 5 minuteincubation at 16° C.

[0368] Phase Lock Gel (Eppendorf, Westbury, N.Y.) andphenol-chloroform-IAA (Invitrogen, Carlsbad, Calif.) were used toextract the cDNA using the manufacturer's protocol. The sample was thenapplied to a MicroCon-30 column (Millipore, Bedford, Mass.) to furtherclean and concentrate the cDNA. The cDNA was dried in a SpeedVac andresuspend in 4.5 μl of RNase-free water.

[0369] III. RNA Amplification

[0370] First round and subsequent rounds of RNA amplification used thecustom designed T3N9 primer (SEQ ID NO: 12) (Invitrogen, Carlsbad,Calif.) for priming the first strand cDNA synthesis. Specifically, 17 μlof first round amplified RNA was mixed with 1 μl of T3N9 (100 pm/μl) andthe mixture was incubated at 70° C. for 10 minutes then chilled, on ice,for 10 minutes. The following reagents were then added to the mixture: 6μl 5×first strand buffer, 1 μl of 10 mM dNTPs (Amersham Pharmacia,Piscataway, N.J.), 3 μl of 0.1 mM DTT (Invitrogen, Carlsbad, Calif.) and2 μl of SuperScript II reverse transcriptase (Invitrogen, Carlsbad,Calif.) were added to the tube, and the reaction mixture was incubatedat 42° C. for 2 hours to carry out first strand cDNA synthesis. Secondstrand cDNA synthesis, double stranded cDNA clean-up and subsequent invitro transcription were performed as described above.

[0371] In addition to the cDNA generated from mRNA, this method yieldeda great deal of cDNA from rRNA and tRNA, however an advantage of thismethod is that it does not favor the synthesis of 3′ products. Moreover,probes labeled after multiple rounds of amplification using templategenerated by this method, behaved well on microarrays (see section VI,below)

[0372] IV. Probe Labeling Using Amine Modified Random Primers

[0373] The amplified RNA can be used as a template for production oflabeled probe molecules using the modified (e.g, amine modified)primers, as described in Examples 4 and 5 above. Five μg of total RNA or2 μg of RNA obtained after three rounds of amplification (5 μg for theamplified RNA obtained directly from cells) were used for labeling thecDNA probes.

[0374] V. cDNA Microarrays

[0375] Amplified RNA generated from 3 rounds of amplification with theT3N9 primer, as described in section III of this Example, above, andtotal RNA were used as templates to generate cDNA probes with the aminemodified random primers, as described in Examples 4 and 5, above. Theprobes were then hybridized to the microarrays as follows: the cDNAprobes were partially dried in a vacuum centrifuge to a volume of 17 μland to the DNA was added 1 μl of poly A (8 mg/ml), 1 μl of Cot-1 DNA (10mg/ml) and 1 μl of yeast tRNA (4 mg/ml). The probe mixture was denaturedat 98° C. for 2 minutes, chilled on ice and 20 μl of the probe mixturewas mixed with 20 μl of 2×F-Hybridization buffer (250 μl of 100%formamide, 250 μl of 20×SSC, 10 μl of 10% sodium dodecyl sulfate). Analiquot of the mixture (35 μl) was applied to arrays. The arrays werecovered with 22×60 mm coverslips and then incubated overnight, in awater bath, at 42° C. Following the incubation, the cover-slips wereremoved from the arrays while they were soaking in pre-wash buffer(2×SSC, 0.1% sodium dodecyl sulfate) and the arrays were washed for 5minutes at room temperature in first wash buffer (0.5×SSC, 0.01% sodiumdodecyl sulfate) followed by a wash with second wash buffer (0.06×SSC)for 5 minutes at room temperature. The arrays were dried by spinningthem in a centrifuge at 800 rpm for 2 minutes.

[0376] All experiments used 41,000 element mouse cDNA arrays. Theexpression profile of a single hypothalamic magnocellular neuron wasidentified.

Example 8 Amplification of DNA Templates With T3N9 Primers Coupled WithAmine Modified Random Priming

[0377] This example describes a method of amplifying a DNA template withthe T3N9 primer, and generating a labeled probe. This method can beused, for instance, to detect a pathogen with a DNA genome, such as thehuman herpes virus 8 (HHV8). The method takes advantage of the fact thatthe reverse transcriptase used is not exclusively an RNA-dependent DNApolymerase. It also has DNA-dependent DNA polymerase activity.Therefore, in the presence of the T3N9 primer, DNA templates are reversetranscribed, and these, in turn, are shown to generate RNA when T3polymerase is added.

[0378] BCBL1 is a human cell line that is latently infected by HHV8.Cellular and viral genomic DNA were isolated from the cells with theQiagen (Valencia, Calif.) genomic DNA isolation kit. The DNA wasserially diluted to 1 μl/μl, 0.1 μg/μl, 0.01 μg/μl and 0.001 μg/μl. Oneμl of each diluted sample was used as a template for RNA amplificationusing the T3N9 primer, as described in Examples 6 and 7, above. RNAamplified from the different sample dilutions was labeled using aminemodified random primers, as described in Examples 5 and 6, above. Theresultant labeled probes were then hybridized to HHV8 arrays imprinted,in duplicate, with DNA corresponding to 88 open reading frames from theHHV8 genome and 100 human house-keeping genes (FIG. 7A).

[0379] In a parallel experiment, PCR amplifications were performed usingthe serially diluted DNA samples described above and a pair of primers(forward primer 5′-TATTCTGCAGCAGCTGTTGG-3′ (SEQ ID NO: 14); reverseprimer 5′-TCTACGTCCAGACGATATGTGC-3′ (SEQ ID NO: 15)) complementary tothe open reading frame sequences of the HHV8 genome. Relatively few DNAspecies were amplified (FIG. 7B).

[0380] Thus, DNA amplification with the T3N9 primers, coupled withamine-modified random priming and microarray detection, is capable ofdetecting a more diverse population of DNAs, compared to the number ofDNA species that can be identified by PCR. Multiple amplification steps,such as the ones described in Examples 6 and 7, above, in combinationwith microarrays can be used to create a method of assaying pathogens inparallel with a sensitivity and specificity better than that of PCR.

Example 9 Fluorescent Nucleotides

[0381] This example describes methods to prepare nucleotides containingat least one fluorophore; such nucleotides may be used as the modifiednucleotide incorporated into modified random primers as disclosedherein. When a the modified nucleotide used to make such random primerscomprises a fluorophore, it is not necessary to react the modifiedprimers, or probes prepared using these primers, with a separatefluorophore (as described for some embodiments above).

[0382] In addition, this example lists some sources of commerciallyavailable fluorescent nucleotides that can be used in the presentdisclosure. Other commercial sources will be known to, or can be readilyascertained by, one of ordinary skill in the art.

[0383] NEN Life Science Products (Boston, Mass.) offers all fourdeoxynucleotides and ribonucleotide analogs with fluorophores attached.There are several different fluorophores available includingfluorescein, Texas Red®, tetramethylrhodamine, coumarin,napthofluorescein, cyanine-3, cyanine-5, and Lissamine™. In addition,Molecular Probes (Eugene, Oreg.) sells deoxyuridinetriphosphate (dUTP)labeled with various fluorophores replacing the methyl group of thymine,synthesized by the method of U.S. Pat. No. 5,047,519. Because thesenucleotides have 3′ hydroxyls, they can be used directly for synthesisreactions.

[0384] Alternatively, nucleotides containing other fluorophores can beprepared. The fluorophores are capable of being attached to thenucleotide, are stable against photobleaching, and have high quantumefficiency. In specific embodiments, the fluorophore does not interfereexcessively with the degree or fidelity of nucleotide incorporation inthe in vitro synthesis reaction used to produce the modified primersdescribed herein. For instance, after attaching a fluorophore, thenucleotide is still able to undergo polymerization, complementary basepairing, and retains a free 3′ hydroxyl end.

[0385] The fluorophore can either be directly or indirectly attached tothe nucleotide, though it is more commonly indirectly attached. Forinstance, the fluorophore may be attached indirectly to the nucleotideby a linker molecule. For example, a streptavidin linkage may be used.

[0386] Alternatively, the modified nucleotide to which the fluorophoreis attached comprises, as part of the modification, a spacer (such as acarbon chain of about 2 to 15 atoms) that links the fluorophore (orreactive group with which the fluorophore reacts) to the nucleotide.U.S. Pat. Nos. 5,047,519 and 5,151,507 to Hobbs et al. (hereinincorporated by reference) teach the use of linkers to separate anucleotide from a fluorophore. Examples of linkers may include astraight or branched chain aliphatic group, particularly a alkyl group,such as C₁-C₂₀, optionally containing within the chain double bonds,triple bonds, aryl groups or heteroatoms such as N, O or S. Substituentson a diradical moiety can include C₁-C₆ alkyl, aryl, ester, ether,amine, amide or chloro groups.

Example 10 Other Uses for Modified Primer Labeling

[0387] Modified primers provided herein can be used in any method thatrequires nucleic acid labeling. The following are examples of knownmethods that incorporate the modified primers provided herein in orderto generate a labeled product.

[0388] Use of Modified Primers in Dendrimer Labeling

[0389] In this example dendrimers, highly branched DNA molecules, arelabeled using a modified primer as provided herein, for example anamine-modified primer. The modified primers contain a sequence in the 5′end that is complementary to a sequence on a dendrimer arm, and thatallows the primer to bind to the dendrimer. The 5′ end of the modifiedprimer also contains a modified base, such as an amino allyl-modifiedbase, to which label detection molecules can be added. Amine modifiedprimers containing amine-modified nucleotides can be synthesized usingin vitro chemical synthesis as is described herein. Examples of labeldetection molecules include, but are not limited to, fluorescentmolecules and biotin. The labeled dendrimers are used, for instance, tohybridize to a cDNA probe. cDNA probes labeled in this manner can beused to generate hybridization signals, for instance in microarrays. Theuse of dendrimers, once they are labeled, is known (see, for example,products and procedures recommended by Genisphere, Hatfield, Pa.).

[0390] Indirect Labeling and Detection of cDNA Using Tyramide SignalAmplification (TSA)

[0391] Tyramide signal amplification (TSA) provides a consistent andreproducible signal amplification method for cDNA microarray analysis.Modified random hexamers, as described herein, for instance withfluorescein or biotin added at one end, can be used as primers tosynthesize labeled cDNA probes from small amounts of total RNA. Purifiedfluorescein and biotin labeled cDNAs are hybridized to microarrays andthe TSA detection method is applied as described in Karsten et al.,Nucleic Acids Research, 30:E4, 2002.

[0392] Labeling RNA Fragments Generated by the DATAS Technique

[0393] Methods can be used to label RNA fragments generated by the DATAStechnique, thereby allowing for a more accurate and sensitivecomparative study of splicing events that characterize distinctphysiopathological situations. RNA fragments generated by the DATAStechnique (Schweighoffer et al., Pharmacogenomics, 1: 187-197, 2000) canbe reverse transcribed or amplified and labeled using amine-modifiedrandom primers that are synthesized as described herein.

Example 11 Other Methods for Labeling Amplified RNA

[0394] RNA amplified by a T3-random primer, such as a T3N9 primer, asdisclosed herein can be used with any RNA or primer labeling method. Thefollowing is an example of a known RNA labeling method that can be usedto add fluorescent dyes directly to RNA.

[0395] Labeling T3N9 Amplified RNA using Platinum Reagents

[0396] RNA amplified by the T3N9 method discussed in Examples 6 and 7,above, can itself be labeled with cyanine fluorophores to generatelabeled probes for microarray experiments. Probes are firmly coupledwith cyanine 3 or cyanine 5 fluorescent dyes by a reactive platinumgroup found in the platinum labeling system ULS (Universal LinkageSystem; Kreatech Biotechnology, The Netherlands). ULS preferentiallyreacts with guanine residues at the N7 position in RNA molecules.Labeled RNA is then purified using simple, column-based protocols,followed by hybridization on a cDNA microarray.

Example 12 Kits For Labeling Probes and Assaying Arrays

[0397] The modified random primers disclosed herein can be supplied inthe form of a kit for use in preparing labeled probes, for instancepreparation of a hybridization probe suitable for assaying a microarray.In specific examples of such kits, an appropriate amount (e.g.,sufficient to prime one or more labeling reactions) of modified randomprimers is provided in one or more containers. The primers may beprovided suspended in an aqueous solution or as a freeze-dried orlyophilized powder, for instance. The container(s) in which the primersare supplied can be any conventional container that is capable ofholding the supplied form, for instance, microfuge tubes, ampoules, orbottles. In some applications, primers may be provided in pre-measuredsingle use amounts in individual, typically disposable, tubes orequivalent containers. With such an arrangement, the sample to belabeled can be added to the individual tubes and reactions carried outdirectly.

[0398] The amount of each primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each random primer provided would likely bean amount sufficient to label several hybridization probes. Those ofordinary skill in the art know the amount of primer that is appropriatefor use in a single labeling reaction; specific examples disclosedherein provide additional guidance.

[0399] In some embodiments of the current invention, kits may alsoinclude the reagents necessary to carry out amplification,polymerization, transcription, or other reactions, including, forinstance, DNA or RNA sample preparation reagents, appropriate buffers(e.g., transcription or polymerase buffer), salts (e.g., magnesiumchloride), deoxyribonucleotides (dNTPs), and/or modified nucleotides(e.g., aa-dUTP).

[0400] Kits may additionally include one or more buffers for use duringassay of an array. For instance, such buffers may include a lowstringency wash, a high stringency wash, and/or a stripping solution.

[0401] Buffers or other constituents provided with kits herein may beprovided in bulk, where each container of is large enough to holdsufficient reagent for several isolation, polymerization, probing,washing, or stripping procedures. Alternatively, the reagents can beprovided in pre-measured aliquots, which might be tailored to the sizeand style of the kit.

[0402] Certain kits may also provide one or more containers in which tocarry out array-probing reactions.

[0403] Kits may in addition include either labeled or unlabeled controlprobe molecules, to provide for internal tests of either the labelingprocedure or probing of an array, or both. The control probe moleculesmay be provided suspended in an aqueous solution or as a freeze-dried orlyophilized powder, for instance. The container(s) in which the controlsare supplied can be any conventional container that is capable ofholding the supplied form, for instance, microfuge tubes, ampoules, orbottles. In some applications, control probes may be provided inpre-measured single use amounts in individual, typically disposable,tubes or equivalent containers.

[0404] The amount of each control probe supplied in the kit can be anyparticular amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, sufficient control probe(s) likely will be provided toperform several controlled analyses of the array. Likewise, wheremultiple control probes are provided in one kit, the specific probesprovided will be tailored to the market and the accompanying kit.

Example 13 Kits for Amplifying Nucleic Acids.

[0405] Components for use in the methods of amplification disclosedherein can be supplied in the form of a kit, for use in amplifying bothDNA and RNA templates, for instance in the preparation of ahybridization probe suitable for assaying a microarray. In specificexamples of such kits, an appropriate amount of T3 random primers (e.g.,sufficient to prime one or more labeling reactions) is provided in oneor more containers. The primers may be provided suspended in an aqueoussolution or as a freeze-dried or lyophilized powder, for instance. Thecontainer(s) in which the primers are supplied can be any conventionalcontainer that is capable of holding the supplied form, for instance,microfuge tubes, ampoules, or bottles. In some applications, primers maybe provided in pre-measured single use amounts in individual, typicallydisposable, tubes or equivalent containers. With such an arrangement,the sample to be amplified can be added to the individual tubes andreactions carried out directly.

[0406] The amount of each primer supplied in the kit can be anyappropriate amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each random primer provided would likely bean amount sufficient to label several nucleic acid templates. Those ofordinary skill in the art know the amount of primer that is appropriatefor use in a single amplification reaction; specific examples disclosedherein provide additional guidance.

[0407] In some embodiments of the current invention, kits may alsoinclude the reagents necessary to carry out the amplification,polymerization, transcription, or other reactions, including, forinstance, DNA or RNA sample preparation reagents, appropriate buffers(e.g., transcription or polymerase buffer), salts (e.g., magnesiumchloride), deoxyribonucleotides (dNTPs), and/or modified nucleotides(e.g., aa-dUTP).

[0408] Buffers or other constituents provided with kits herein may beprovided in bulk, where each container of is large enough to holdsufficient reagent for several isolation, polymerization, probing,washing, or stripping procedures. Alternatively, the reagents can beprovided in pre-measured aliquots, which might be tailored to the sizeand style of the kit.

[0409] Certain kits may also provide one or more containers in which tocarry out labeling reactions.

[0410] Kits may in addition include either labeled or unlabeled controlmolecules, such as a known amount of nucleic acid template, to providefor internal tests of either the amplification procedure or labelingprocedure, or both. The control molecules may be provided suspended inan aqueous solution or as a freeze-dried or lyophilized powder, forinstance. The container(s) in which the controls are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, or bottles. In some applications,control molecules may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers.

[0411] The amount of each control molecule supplied in the kit can beany particular amount, depending for instance on the market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, sufficient control molecules likely will be provided toperform several controlled analyses of the array. Likewise, wheremultiple control molecules are provided in one kit, the specificmolecules provided will be tailored to the market and the accompanyingkit.

[0412] This disclosure provides methods of amplifying nucleic acidtemplates and methods of producing modified nucleic acid molecules,including labeled nucleic acids, for use in hybridization reactions,using modified random primers to initiate synthesis. The disclosurefurther provides modified random primers, modified probe nucleic acidmolecules produced by methods disclosed herein, and methods of usingthese molecules. It will be apparent that the precise details of themethods and compositions described may be varied or modified withoutdeparting from the spirit of the described invention. All suchmodifications and variations that fall within the scope and spirit ofthe claims below are claimed.

1 15 1 7 DNA artificial sequence synthetic oligonucleotide 1 nnnnnnn 7 26 DNA artificial sequence synthetic oligonucleotide 2 nnnnnn 6 3 8 DNAartificial sequence synthetic oligonucleotide 3 nnnnnnnn 8 4 9 DNAartificial sequence synthetic oligonucleotide 4 nnnnnnnnn 9 5 10 DNAartificial sequence synthetic oligonucleotide 5 nnnnnnnnnn 10 6 11 DNAartificial sequence synthetic oligonucleotide 6 nnnnnnnnnn n 11 7 11 DNAartificial sequence synthetic oligonucleotide 7 nnnnnnnnnn n 11 8 13 DNAartificial sequence synthetic oligonucleotide 8 nnnnnnnnnn nnn 13 9 14DNA artificial sequence synthetic oligonucleotide 9 nnnnnnnnnn nnnn 1410 7 DNA artificial sequence synthetic oligonucleotide 10 nnnnnnn 7 1155 DNA artificial sequence synthetic oligonucleotide 11 aaacgacggccagtgaattg taatacgact cactataggc gctttttttt ttttt 55 12 41 DNAartificial sequence synthetic oligonucleotide 12 gcgcgaaatt aaccctcactaaagggagag ggnnnnnnnn n 41 13 59 DNA artificial sequence syntheticoligonucleotide 13 ggccagtgaa ttgtaatacg actcactata gggaggcggttttttttttt ttttttttt 59 14 20 DNA artificial sequence syntheticoligonucleotide 14 tattctgcag cagctgttgg 20 15 22 DNA artificialsequence synthetic oligonucleotide 15 tctacgtcca gacgatatgt gc 22

What is claimed is:
 1. A method of producing a modified nucleic acidprobe, comprising: contacting a nucleic acid template with a modifiedrandom primer under conditions sufficient to permit base-specifichybridization between the template and the primer, wherein the modifiedrandom oligonucleotide primer comprises an amine-modified dNTP or alabel-substituted dNTP; and polymerizing a nucleic acid moleculecomplementary to a nucleic acid sequence in the template andincorporating at least one modified oligonucleotide primer, therebyproducing the modified nucleic acid probe.
 2. The method of claim 1,wherein the modified random primer is modified at the five prime end ofthe primer.
 3. The method of claim 1, wherein the modified random primercomprises an amine-modified dNTP, the method further comprising:coupling the modified nucleic acid probe to a label molecule to form alabel-probe conjugate.
 4. A modified random primer for use in the methodof claim
 1. 5. The modified primer of claim 4, wherein the primer is anyone of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ IDNO:
 10. 6. The modified random primer of claim 4, wherein the primer isP2 (SEQ ID NO: 1).
 7. The modified random primer of claim 4, wherein theprimer is P4 (SEQ ID NO: 2).
 8. The method of claim 1, wherein thenucleic acid template comprises a mixture of nucleic acid molecules. 9.The method of claim 8, wherein the mixture of nucleic acid moleculescomprises RNA.
 10. The method of claim 9, wherein polymerizing comprisespolymerizing a cDNA.
 11. A method of producing a fluorescenthybridization probe, comprising: contacting a template nucleic acidsample with a modified random primer comprising at least one aminoallyldUTP residue; polymerizing a nucleic acid molecule complementary to asequence in the template sample and incorporating one or more modifiedrandom primers, to produce a modified complementary nucleotide; andcontacting the modified complementary nucleotide with an amine-reactivefluorescent label, thereby producing the fluorescent hybridizationprobe.
 12. The method of claim 11, wherein aminoallyl dNTP is includedduring polymerizing.
 13. The method of claim 11, wherein the templatenucleic acid comprises mRNA and polymerizing comprises reversetranscription.
 14. A fluorescent hybridization probe produced by themethod of claim
 11. 15. An improved method for random primer reversetranscription labeling of a nucleic acid hybridization probe, theimprovement comprising using random primers modified with at least oneamine-substituted dNTP or fluorescent-dye modified dNTP in the reversetranscription reaction.
 16. An improved hybridization probe as producedby the method of claim
 15. 17. The method of claim 1, wherein thenucleic acid template is an amplified nucleic acid template.
 18. A kitfor producing a labeled hybridization probe or for probing an array,comprising the modified random primer of claim
 4. 19. The method ofclaim 1, wherein the nucleic acid template is originally isolated from asmall number of cells.
 20. The method of claim 19, wherein the smallnumber of cells is lysed by sonication in a buffer comprising firststrand buffer and an RNase inhibitor.
 21. The method of claim 19,wherein the small number of cells is less than about 1000 cells.
 22. Themethod of claim 19, wherein the small number of cells is less than about100 cells.
 23. The method of claim 19, wherein the small number of cellsis about 10 cells.
 24. The method of claim 19, wherein the small numberof cells is about 1 cell.
 25. The method of claim 19, wherein thenucleic acid template is an amplified template.
 26. The method of claim25, wherein the amplified template comprises RNA.
 27. The method ofclaim 26, further comprising contacting the amplified template with asecond primer, wherein the second primer has a nucleic acid sequence asset forth in SEQ ID NO: 12, under conditions sufficient to permitbase-specific hybridization between the template and the second primer.28. The method of claim 27, wherein the second primer, comprising anucleic acid sequence as set forth in SEQ ID NO: 12, is used in at leastone round of cDNA synthesis other than the first round.
 29. The methodof claim 27, wherein the modified random primer comprises anamine-modified dNTP, the method further comprises coupling theamine-modified nucleic acid probe to a label molecule to form alabel-probe conjugate.
 30. A method of producing an RNA template from asmall number of cells, comprising: lysing a small number of cells bysonication in a buffer, wherein the buffer comprises first strand bufferand an RNase inhibitor, to produce a lysate, wherein the lysatecomprises the RNA nucleic acid template.
 31. The method of claim 30,wherein the small number of cells comprises less than about ten cells.32. The method of claim 30, wherein the small number of cells comprisesabout one cell.
 33. A method of producing a modified nucleic acid probe,comprising: amplifying the RNA template of claim 30 to produce anamplified template; generating cDNA from the amplified template;contacting the cDNA with a modified random primer comprising anamine-modified dNTP under conditions sufficient to permit hybridizationbetween the cDNA and the modified random primer; and polymerizing anucleic acid molecule complementary to a nucleic acid sequence in thecDNA and incorporating at least one modified oligonucleotide primer,thereby producing the modified nucleic acid probe.
 34. The method ofclaim 1, wherein a second primer, comprising a nucleic acid sequence asset forth in SEQ ID NO: 12, contacts the nucleic acid template underconditions sufficient to permit base-specific hybridization between thetemplate and the second primer and generates an amplified nucleic acidtemplate that is capable of hybridizing with the modified random primer.35. The method of claim 34, wherein the nucleic acid template comprisesa mixture of nucleic acid molecules.
 36. The method of claim 35, whereinthe mixture of nucleic acid molecules comprises RNA.
 37. The method ofclaim 36, wherein the RNA comprises ribosomal RNA, messenger RNA,transfer RNA, or mixtures thereof.
 38. The method of claim 34, whereinthe template is derived from a cell or a virus.
 39. The method of claim35, wherein the mixture of nucleic acid molecules comprises DNA.
 40. Themethod of claim 34, wherein the nucleic acid template is isolated from asmall number of cells.
 41. The method of claim 40, wherein the smallnumber of cells is less than about 1000 cells.
 42. The method of claim40, wherein the small number of cells is less than about 100 cells. 43.The method of claim 40, wherein the small number of cells is about 10cells.
 44. The method of claim 40, wherein the small number of cells isabout 1 cell.
 45. The method of claim 40, wherein the small number ofcells is 1 cell.
 46. The method of claim 40, wherein the small number ofcells are infected with a virus.
 47. The method of claim 46, wherein thevirus is a DNA virus or an RNA virus.
 48. The method of claim 47,wherein the virus is human herpes virus-8.
 49. The method of claim 34,wherein the second primer comprising a nucleic acid sequence as setforth in SEQ ID NO: 12 is used in at least one round of cDNA synthesis.50. The method of claim 34, wherein the modified random primer ismodified at the five prime end of the primer.
 51. The method of claim34, further comprising coupling the modified nucleic acid probe to alabel molecule to form a label-probe conjugate.
 52. The method of claim34, wherein the modified random primer is any one of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:
 10. 53. A method ofamplifying a nucleic acid template, comprising: contacting a nucleicacid template with a primer under conditions sufficient to permitbase-specific hybridization between the template and the primer, whereinthe primer comprises a T3-promoter and a random primer and wherein therandom primer comprises between about 4 and about 12 nucleotides;polymerizing a nucleic acid molecule complementary to a nucleic acidsequence in the template, to produce a polymerized nucleic acidmolecule; and amplifying the polymerized nucleic acid molecule, therebyamplifying a nucleic acid template.
 54. The method of claim 53, whereinthe primer comprises a T3N9 primer with a sequence as set forth in SEQID NO:
 12. 55. The method of claim 53, further comprising coupling theamplified nucleic acid template to a label molecule.
 56. The method ofclaim 55, wherein the label molecule is a fluorophore or a hapten. 57.The method of claim 55, wherein labeling the amplified nucleic acidtemplate comprises contacting the amplified nucleic acid template with amodified random primer comprising at least one aminoallyl dNTP residue;polymerizing a nucleic acid molecule complementary to a sequence in theamplified nucleic acid template and incorporating one or more modifiedrandom primers, to produce a modified complementary nucleotide; andcontacting the modified complementary nucleotide with an amine-reactivefluorescent label, thereby producing the labeled amplified nucleic acidtemplate.